Novel phosphodiesterase inhibitors and uses thereof

ABSTRACT

The invention provides for novel benzonaphthyridine derivatives and compositions comprising novel benzonaphthyridine derivatives. In some embodiments, the compounds are phosphodiesterase inhibitors. The invention further provides for methods for inhibition of phosphodiesterase comprising contacting phosphodiesterase with novel benzonaphthyridine derivatives or compositions comprising novel benzonaphthyridine derivatives. The invention further provides for methods for treatment of neurodegenerative diseases, increasing memory or long term potentiation with novel benzonaphthyridine derivatives or compositions comprising novel benzonaphthyridine derivatives. In some embodiments, the phosphodiesterase is PDE5.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 61/847,280, filed on Jul. 17, 2013, the contentof which is hereby incorporated by reference in its entirety.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

GOVERNMENT SUPPORT

This invention was made with government support under NIH/NIA Grant No.1U01AG032973 awarded by the National Institute of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disorder that is themost common cause of dementia among elderly persons. It is characterizedby progressive memory loss, synaptic dysfunction and accumulation ofamyloid β-peptides (Aβ). It is caused in part by increased levels ofamyloid-β-peptide 1-42 (Aβ42). Approved drugs to treat AD includecholinesterase inhibitors such as Cognex® (tacrine), Aricept®(donepezil), Exelon® (rivastigmine) and Razdyne® (galantamine); and theN-methyl d-aspartate receptor antagonist Namenda® (memantine). However,several of these medications suffer from limited efficacy and produceuntoward side effects.

Phosphodiesterase 5 (PDE5) inhibitors are widely used as therapeuticsfor erectile dysfunction and pulmonary hypertension. These inhibitorsare believed to increase cGMP levels, which enhances phosphorylation ofthe transcription factor and memory-affecting molecule cAMP-responsiveelement binding (CREB) through activation of cGMP-dependent-proteinkinases.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate(cGMP), nucleotide biological second messengers, regulate variousbiological processes, such as blood flow regulation, cardiac musclecontraction, cell differentiation, neural transmission, glandularsecretion, and gene expression. Intracellular receptors for thesemolecules include cyclic nucleotide phosphodiesterases (PDEs), cyclicnucleotide dependent protein kinases (PGK), and cyclic nucleotide-gatedchannels. PDEs are a large family of proteins that catalyze thehydrolysis of 3′,5′-cyclic nucleotides to the corresponding 5′monophosphates. There are eleven related, but biochemically distinct,human PDE gene groups. Some PDEs are specific for hydrolysis of cAMP(such as PDE4, PDE7, and PDE8), and some are cGMP specific (such asPDE5, PDE6, and PDE9), while some PDEs have mixed specificity (such asPDE1, PDE2, PDE3, PDE10, and PDE11).

Representative PDE 5 inhibitors are cyclic guanosine 3′,5′-monophosphatetype five cGMP PDE inhibitors, also known as PDE-5 inhibitors, whichinclude, for example, sildenafil, tadalafil, zaprinast, and vardenafil.PDE5 inhibitors increase cGMP levels by inhibiting the degradativeaction of PDE5 on cGMP. Current PDE5 inhibitors suffer from drawbackssuch as limited selectivity over other PDE sub-types. There remains aneed for structurally novel PDE5 inhibitors.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a class ofbenzo[b][1,6]naphthyridine derivatives of formula (I)

wherein

A is O or NR⁴; B is CR¹⁶ or N;

V is a bond or C(O);W is a bond or NR¹³;X is —(C₁-C₃)-alkyl, —(C1

C3)-alkyl substituted with at least one D, C(O), S, S(O), or S(O)₂;

Y is NR⁵, O or S; Z is C or N;

R¹ is hydrogen, halogen or —(C₁-C₆)-haloalkyl;R² is hydrogen or —OR⁶;R³ is hydrogen, —OMe, —CF₃, —CN or halogen;R⁴ is hydrogen or —(C₁-C₃)-alkyl;R⁵ is hydrogen, —(C₁-C₃)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, —S(O)₂R⁷;R⁶ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or—(C₃-C₈)-cycloalkyl;R⁷ is independently hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, oraryl;R⁸ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, —(C₃-C₈)-cycloalkyl,—NR⁹R¹⁰, —S(O)₂R¹¹, orheterocyclyl;R⁹ and R¹⁰ are each independently hydrogen, —(C₁-C₆)-alkyl,—(C₃-C₈)-cycloalkyl, or C(O)R¹¹, wherein the —(C₁-C₆)-alkyl or—(C₃-C₈)-cycloalkyl are optionally substituted with —(C₁-C₆)-alkyl,—(C₃-C₈)-cycloalkyl, —NR¹¹R¹², —SR¹¹, K or heterocyclyl; or, R⁹ and R¹⁰together with the nitrogen atom to which they are attached form a 3 to8-membered heterocycle, wherein any one of the ring carbon atoms isoptionally replaced with a heteroatom, and wherein the heterocycle isoptionally substituted with —(C₁-C₆)-alkyl;R¹¹ and R¹² are each independently hydrogen, —(C₁-C₆)-alkyl, or—(C₃-C₈)-cycloalkyl;R¹³ is hydrogen, —(C₁-C₆)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, or —S(O)₂R⁷;R¹⁴ is hydrogen, halogen or —(C₁-C₆)-haloalkyl;R¹⁵ is hydrogen, —OR¹⁷, OH or halogen;R¹⁶ is hydrogen, —OR¹⁷, OH or halogen;R¹⁷ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or—(C₃-C₈)-cycloalkyl;m and n are each 0, 1, 2, or 3, provided that the sum of m+n is aninteger from 2-4;or a pharmaceutically acceptable salt or tautomer thereof; with theproviso that R¹ and R² are not both hydrogen when V and W are each abond, Y is NR⁵, A is NR⁴, X is CO, n=2 and m=1.

In some embodiments, the compounds of the invention decrease PDEactivity by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%, or 100%.

In some embodiments, the compounds of the invention inhibitphosphodiesterase. In some embodiments, the phosphodiesterase isphosphodiesterase type V (PDE5).

In some embodiments, the compounds of the invention have an IC50 forphosphodiesterase of at least about 0.1 nM, at least about 1 nM, atleast about 5 nM, at least about 10 nM, at least about 25 nM, at leastabout 50 nM, at least about 100 nM, at least about 200 nM, at leastabout 300 nM, at least about 400 nM, at least about 500 nM, at leastabout 600 nM, at least about 700 nM, at least about 800 nM, at leastabout 900 nM, or at least about 1000 nM.

In another aspect, the invention is directed to compositions comprisinga compound of formula (I) and a pharmaceutically acceptable carrier.

In another aspect, the invention is directed to a method of inhibitingphosphodiesterase comprising contacting a phosphodiesterase with acompound of formula (I) or a composition comprising a compound offormula (I). In some embodiments, the phosphodiesterase is PDE5.

In another aspect, the invention is directed to a method of treatingneurodegenerative disease in a subject comprising administration of atherapeutically effective amount of a compound of formula (I). In someembodiments, the disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of increasinglong-term potentiation in a subject comprising administration of atherapeutically effective amount of a compound of formula (I).

In another aspect, the invention is directed to a method of improvingmemory in a subject comprising administration of a therapeuticallyeffective amount of a compound of formula (I). In some embodiments, thesubject has a neurodegenerative disease. In some embodiments, theneurodegenerative disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject comprising administration of atherapeutically effective amount of a compound of formula (I). In someembodiments, the subject has a neurodegenerative disease. In someembodiments, the neurodegenerative disease is Alzheimer's Disease.

Still other objects and advantages of the invention will become apparentto those of skill in the art from the disclosure herein, which is simplyillustrative and not restrictive. Thus, other embodiments will berecognized by the skilled artisan without departing from the spirit andscope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effects of compound H on hippocampal cGMP levels inmice.

FIG. 2 shows the effects of compound H in wild-type and APP/PS1 mice, atransgenic mouse model of AD, on residual potentiation recorded duringthe last 5 minutes of a 2 hr recording following tetanic stimulation ofthe Schaffer collateral fibers at the CA3-CA1 connection.

FIG. 3 shows the effects of compound H in wild-type and APP/PS1 mice, atransgenic mouse model of AD, on residual potentiation recorded duringthe last 5 minutes of a 2 hr recording following tetanic stimulation ofthe Schaffer collateral fibers at the CA3-CA1 connection. Dailytreatment with compound H (3 mg/kg, i.p.) for 3 weeks at the age of 3-4months re-established normal potentiation when slices were recorded at6-7 months of age.

FIG. 4 shows the effects of compound H on defects in reference memory inwild-type and APP/PS1 mice, a transgenic mouse model of AD, in the 2 dayradial arm water maze. Daily treatment with the compound for 3 weeks atthe age of 3-4 months reduced the number of errors with the 2-day radialarm water maze in APP/PS1 mice.

FIG. 5 shows the effects of compound H on defects in fear memory inwild-type and APP/PS1 mice, a transgenic mouse model of AD, oncontextual fear memory. Daily treatment with the compound for 3 weeks atthe age of 3-4 months re-established normal freezing in a test forcontextual fear memory in APP/PS1 mice.

FIG. 6 shows the effects of compound H on defects in reference memory inwild-type and APP/PS1 mice, a transgenic mouse model of AD, in the 2 dayradial arm water maze. Daily treatment with compound (3 mg/kg, i.p.) for3 weeks at the age of 3-4 months reduced the number of errors with the2-day radial arm water maze when mice were examined at 6-7 months ofage.

FIG. 7 shows the effects of compound H on defects in fear memory inwild-type and APP/PS1 mice, a transgenic mouse model of AD, oncontextual fear memory. Daily treatment with compound (3 mg/kg, i.p.)for 3 weeks at the age of 3-4 months re-established normal freezing in atest for contextual fear memory when mice were examined at 6-7 months ofage.

FIG. 8 shows concentrations-time course of compound H in mice brain andplasma following i.p. administration of 25 mg/kg dose.

FIG. 9 shows H&E staining for three groups (A: vehicle; B: 120 mg/kgcompound H; C: 240 mg/kg compound H); and a GFAP immunostain for threegroups (D: vehicle; E: 120 mg/kg compound H, F: 240 mg/kg compound H).

DETAILED DESCRIPTION OF THE INVENTION

Treatment for AD remains a major focus in the medical community. To thatend, several biological targets for treatment of AD are being exploredsuch as Tau, beta-secretase, and gamma-secretase. AD purportedly beginsas a synaptic disorder produced at least in part, by Aβ (Selkoe, D. J.Science 2002, 298, 789-791; herein incorporated by reference in itsentirety). Aβ-induced reduction in long-term-potentiation (LTP), aphysiological correlate of synaptic plasticity that is thought tounderlie learning and memory, and phosphorylation of the memorytranscription factor CREB, are ameliorated by nitric oxide (NO) donorsand cGMP-analogs (Puzzo, et al, J. Neurosci 2005, 25, 6887-6897; hereinincorporated by reference in its entirety). Vice-versa, genetic ablationof NO-synthase 2 (NOS2) results in worsening of the AD phenotype in miceexpressing mutated amyloid precursor protein (APP) (Colton et al.Proceedings of the National Academy of Sciences of the United States ofAmerica 2006, 103, 12867-12872; herein incorporated by reference in itsentirety). Taken together, these findings show that up-regulation of theNO pathway can be protective in AD.

Despite the neuroprotective function of NO, the gas has also been viewedas a major agent of neuropathology and cell death when produced in highquantities. High amounts of NO lead to generation of significantquantity of peroxinitrites that are responsible for oxidative andnitrosative stress in Aβ-induced cell death. Release of low amounts ofNO by the constitutive forms of NOS that include both the neuronal andthe endothelial isoforms, n-NOS and e-NOS, promotes synaptic plasticityand learning, whereas uncontrolled production of high amounts of the gasby the inducible form of NOS (i-NOS) can promote oxidative andnitrosative stress via production of peroxinitrite. Thus, bothAβ-induced downregulation of the NO cascade which blocks plasticity andmemory and generation of peroxinitrites leading to cell death, can playroles in AD.

Strategies that can bypass NO production focus on steps downstream of NOgeneration. Agents that enhance NO/cGMP/CREB signaling can rescueAb-induced reduction of synaptic plasticity and memory (See, WO2010/074783 and references cited therein; Prickaerts, et al., Eur. J.Pharmacol. 1999, 10, 731-737; and Neuroscience 2002, 113, 351-361; eachherein incorporated by reference in its entirety).

In one aspect, the invention is directed to derivatives of formula (I):

more particularly to a derivatives of formula (Ia):

still more particularly to derivatives of formula (Ib):

wherein

A is O or NR⁴; B is CR¹⁶ or N;

V is a bond or C(O);W is a bond or NR¹³;X is —(C₁-C₃)-alkyl, —(C1

C3)-alkyl substituted with at least one D, C(O), S, S(O), or S(O)₂;

Y is NR⁵, O or S; Z is C or N;

R1 is hydrogen, halogen or —(C1-C6)-haloalkyl;R² is hydrogen or —OR⁶;R3 is hydrogen, —OMe, —CF3, —CN or halogen;R⁴ is hydrogen or —(C₁-C₃)-alkyl;R⁵ is hydrogen, —(C₁-C₃)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, —S(O)₂R⁷;R⁶ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or—(C₃-C₈)-cycloalkyl;R⁷ is independently hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, oraryl;R⁸ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, —(C₃-C₈)-cycloalkyl,—NR⁹R¹⁰, —S(O)₂R¹¹, or heterocyclyl;R⁹ and R¹⁰ are each independently hydrogen, —(C₁-C₆)-alkyl,—(C₃-C₈)-cycloalkyl, or C(O)R¹¹, wherein the —(C₁-C₆)-alkyl or—(C₃-C₈)-cycloalkyl are optionally substituted with —(C₁-C₆)-alkyl,—(C₃-C₈)-cycloalkyl, —NR¹¹R¹², —S¹¹, or heterocyclyl; or, R⁹ and R¹⁰together with the nitrogen atom to which they are attached form a 3 to8-membered heterocycle, wherein any one of the ring carbon atoms isoptionally replaced with a heteroatom, and wherein the heterocycle isoptionally substituted with —(C₁-C₆)-alkyl;R¹¹ and R¹² are each independently hydrogen, —(C₁-C₆)-alkyl, or—(C₃-C₈)-cycloalkyl;R¹³ is hydrogen, —(C₁-C₆)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, or —S(O)₂R⁷;R¹⁴ is hydrogen, halogen or —(C₁-C₆)-haloalkyl;R¹⁵ is hydrogen, —OR¹⁷, —OH or halogen;R¹⁶ is hydrogen, —OR¹⁷, OH or halogen;R¹⁷ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or—(C₃-C₈)-cycloalkyl;m and n are each 0, 1, 2, or 3, provided that the sum of m+n is aninteger from 1-4;or a pharmaceutically acceptable salt or tautomer thereof; with theproviso that R¹ and R² are not both hydrogen when V and W are each abond, Y is NR⁵, A is NR⁴, X is CO, n=2 and m=1.

In another aspect, the invention is directed to derivatives of formula(Ic):

wherein

A is O or NR⁴; B is CR¹⁶ or N;

V is a bond or C(O);W is a bond or NR¹³;X is —(C₁-C₃)-alkyl, —(C1

C3)-alkyl substituted with at least one D, C(O), S, S(O), or S(O)₂;

Y is NR⁵, O or S;

R¹ is hydrogen, halogen or —(C₁-C₆)-haloalkyl;R² is hydrogen or OR⁶;R³ is hydrogen, —OMe, —CF₃, —CN or halogen;R⁴ is hydrogen or —(C₁-C₃)-alkyl;R⁵ is hydrogen, —(C₁-C₃)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, —S(O)₂R⁷;R⁶ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or—(C₃-C₈)-cycloalkyl;R⁷ is independently hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, oraryl;R⁸ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, —(C₃-C₈)-cycloalkyl,—NR⁹R¹⁰, —S(O)₂R¹¹, or heterocyclyl;R⁹ and R¹⁰ are each independently hydrogen, —(C₁-C₆)-alkyl,—(C₃-C₈)-cycloalkyl, or C(O)R¹¹, wherein the —(C₁-C₆)-alkyl or—(C₃-C₈)-cycloalkyl are optionally substituted with —(C₁-C₆)-alkyl,—(C₃-C₈)-cycloalkyl, —NR¹¹R¹², —S¹¹, or heterocyclyl; or, R⁹ and R¹⁰together with the nitrogen atom to which they are attached form a 3 to8-membered heterocycle, wherein any one of the ring carbon atoms isoptionally replaced with a heteroatom, and wherein the heterocycle isoptionally substituted with —(C₁-C₆)-alkyl;R¹¹ and R¹² are each independently hydrogen, —(C₁-C₆)-alkyl, or—(C₃-C₈)-cycloalkyl;R¹³ is hydrogen, —(C₁-C₆)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, or —S(O)₂R⁷;R¹⁵ is hydrogen, —OR¹⁷, —OH or halogen;R¹⁶ is hydrogen, —OR¹⁷, —OH or halogen;R¹⁷ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or—(C₃-C₈)-cycloalkyl;m and n are each 0, 1, 2, or 3, provided that the sum of m+n is aninteger from 1-4;or a pharmaceutically acceptable salt or tautomer thereof; with theproviso that R¹ and R² are not both hydrogen when V and W are each abond, Y is NR⁵, A is NR⁴, X is CO, n=2 and m=1.

The compounds and/or compositions of the invention may be effective intreating, reducing, and/or suppressing complications related to PDE,including PDE5, such as, for example, erectile dysfunction.

ABBREVIATIONS AND DEFINITIONS

The terms “phosphodiesterase inhibitor” or “PDE inhibitor” refer tocompounds and salts or solvates thereof that function by inhibiting theactivity of the enzyme phosphodiesterase. An exemplary phosphodiesteraseis phosphodiesterase type 5 (PDE5). A PDE inhibitor can be a compoundthat decreases the activity of PDE in vivo and/or in vitro. ExemplaryPDE5 inhibitors may be found in U.S. Pat. Nos. 5,250,534; 5,859,006;6,362,178; and 7,378,430; each of which hereby incorporated by referencein its entirety.

The term “compound of the invention” as used herein means a compound offormula (I) or any subgenus or species thereof. The term is alsointended to encompass salts, hydrates, and solvates thereof

The term “composition(s) of the invention” as used herein meanscompositions comprising a compound of the invention, and salts,hydrates, or solvates thereof. The compositions of the invention mayfurther comprise other agents such as, for example, carriers,excipients, stabilants, lubricants, solvents, and the like.

The term “alkyl”, as used herein, unless otherwise indicated, refers toa monovalent aliphatic hydrocarbon radical having a straight chain orbranched chain. Examples of “alkyl” groups include methyl, ethyl,propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl,hexyl, heptyl, and the like.

The term “D” refers to a deuterium atom, and is known in the art torefer to a deuterium enriched species, that is, where D is present aboveits natural isotopic abundance.

The term “haloalkyl”, as used herein, refers to an alkyl groupsubstituted with one or more halogen atoms. The term also encompasseshaloalkyl groups containing more than one species of halogen atom, forexample —CF₂Cl, and the like.

The term “halogen”, as used herein, means chlorine (Cl), fluorine (F),iodine (I) or bromine (Br).

The term “solvate” as used herein means a compound, or apharmaceutically acceptable salt thereof, wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent is physiologically tolerable at the dosage administered.Examples of suitable solvents are ethanol, water and the like. Whenwater is the solvent, the molecule is referred to as a “hydrate.”

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or pharmaceutically acceptable salts,solvates, or hydrates thereof, with other chemical components, such asphysiologically acceptable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of a compoundto an organism or subject.

The term “pharmaceutically acceptable salt” is intended to include saltsderived from inorganic or organic acids including, for examplehydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric,formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic,salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic,trifluroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids;and salts derived from inorganic or organic bases including, for examplesodium, potassium, calcium, ammonium or tetrafluoroborate. Exemplarypharmaceutically acceptable salts are found, for example, in Berge, etal. (J. Pharm. Sci. 1977, 66(1), 1; hereby incorporated by reference inits entirety).

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Non-limiting examples of suchpharmaceutical carriers include liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical carriers may also be saline, gum acacia, gelatin,starch paste, talc, keratin, colloidal silica, urea, and the like. Inaddition, auxiliary, stabilizing, thickening, lubricating and coloringagents may be used. Other examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences (Alfonso Gennaroed., Krieger Publishing Company (1997); Remington's: The Science andPractice of Pharmacy, 21^(st) Ed. (Lippincot, Williams & Wilkins (2005);Modern Pharmaceutics, vol. 121 (Gilbert Banker and Christopher Rhodes,CRC Press (2002); each of which hereby incorporated by reference in itsentirety).

PDE5 inhibitors are described, for example, in U.S. Pat. Nos. 5,250,534;5,859,006; 6,362,178; and 7,378,430; International Patent PublicationNos. WO/2008/095835, WO/2009/050554, WO/2009/124119, WO/2010/015589,WO/2010/074783, and WO/2011/015523; and Uthayathas et al in Pharmacol.Rep. 2007, 59(2), 150-63; and references cited therein; each of which ishereby incorporated by reference in its entirety. PDE5 inhibitorsinclude, for example, sildenafil, tadalafil, vardenafil, avanafil,lodenafil, udenafil, mirodenafil, P20066 (Ethypharm), SLx-2101 (KadmonPharmaceuticals), PF00489791 (Pfizer), INT007 (IntelGenx Technologies),and dasantafil. A novel family of PDE5 inhibitors has been discoveredand is described herein.

Benzonaphthyridine derivatives are described, for example, in U.S. Pat.Nos. 3,674,790; 4,742,061; 6,294,547; 6,384,047; 6,436,952; andInternational Patent Publication No. WO/1998/055481; each of which ishereby incorporated by reference in its entirety. Herein, the inventorsdescribe compounds that are novel benzonaphthyridine derivatives. Insome embodiments, the compounds inhibit PDE5.

In some embodiments, A is O. In some embodiments, A is NR⁴. In someembodiments, A is N—(C₁-C₃)-alkyl. In some embodiments, A is N-methyl.In some embodiments, A is NH.

In some embodiments, X is —(C₁-C₃)-alkyl. In some embodiments, X is—(C₁-C₂)-alkyl. In some embodiments, X is CH₂.

In some embodiments, V is a bond. In some embodiments, V is C(O).

In some embodiments, W is a bond. In some embodiments, W is NR¹³.

In some embodiments, R¹ is hydrogen or halogen. In some embodiments, R¹is hydrogen, halogen or —(C₁-C₃)-haloalkyl. In some embodiments, R¹ ishalogen or —(C₁-C₆)-haloalkyl. In some embodiments, R¹ is halogen or—(C₁-C₃)-haloalkyl. In some embodiments, R¹ is halogen. In someembodiments, R¹ is fluorine, chlorine or bromine. In some embodiments,R¹ is chlorine or bromine. In some embodiments, R¹ is chlorine. In someembodiments, R¹ is —(C₁-C₃)-haloalkyl. In some embodiments, R¹ istrifluoromethyl.

In some embodiments, R² is OR⁶. In some embodiments, R² is—O—(C₁-C₃)-alkyl. In some embodiments, R² is —O-methyl or —O-ethyl. Insome embodiments, R² is —O-methyl. In some embodiments, R² is hydrogen.

In some embodiments, R³ is hydrogen, —OMe, —CF₃, —CN or halogen. In someembodiments, R³ is CN. In some embodiments, R³ is halogen. In someembodiments, R³ is fluorine, chlorine or bromine. In some embodiments,R³ is fluorine. In some embodiments, R³ is chlorine. In someembodiments, R³ is bromine. In some embodiments, R³ is hydrogen. Inother embodiments, R³ is —OMe. In some embodiments, R³ is —CF₃.

In some embodiments, R⁵ is hydrogen, —(C₁-C₃)-alkyl, or—(C₃-C₅)-cycloalkyl. In some embodiments, R⁵ is hydrogen or—(C₁-C₃)-alkyl. In some embodiments, R⁵ is —(C₁-C₃)-alkyl or—(C₃-C₅)-cycloalkyl. In some embodiments, R⁵ is hydrogen. In someembodiments, R⁵ is —(C₁-C₃)-alkyl. In some embodiments, R⁵ is methyl orethyl. In some embodiments, R⁵ is methyl. In some embodiments, R⁵ isethyl. In some embodiments, R⁵ is —(C₃-C₅)-cycloalkyl. In someembodiments, R⁵ is cyclopropyl or cyclobutyl. In some embodiments, R⁵ iscyclopropyl.

In some embodiments, R⁶ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl,or —(C₃-C₈)-cycloalkyl. In some embodiments, R⁶ is hydrogen,—(C₁-C₆)-alkyl, or —(C₁-C₆)-haloalkyl. In some embodiments, R⁶ ishydrogen, —(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl. In some embodiments, R⁶is hydrogen, or —(C₁-C₃)-alkyl. In some embodiments, R⁶ is—(C₁-C₃)-alkyl.

In some embodiments, R⁷ is independently hydrogen, —(C₁-C₆)-alkyl,—(C₁-C₆)-haloalkyl, or aryl. In some embodiments, R⁷ is independentlyhydrogen, —(C₁-C₆)-alkyl, or —(C₁-C₆)-haloalkyl. In some embodiments, R⁷is independently hydrogen or —(C₁-C₆)-alkyl.

In some embodiments, R⁸ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl,—(C₃-C₈)-cycloalkyl, —NR⁹R¹⁰, —S(O)₂R¹¹, or heterocyclyl. In someembodiments, R⁸ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl,—(C₃-C₈)-cycloalkyl, or —NR⁹R¹⁰. In some embodiments, R⁸ is hydrogen,—(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or —(C₃-C₈)-cycloalkyl. In someembodiments, R⁸ is hydrogen, —(C₁-C₆)-alkyl, or —(C₃-C₈)-cycloalkyl. Insome embodiments, R⁸ is hydrogen or —(C₁-C₆)-alkyl. In some embodiments,R⁸ is hydrogen or —(C₁-C₃)-alkyl. In some embodiments, R⁸ is hydrogen or—(C₁-C₂)-alkyl. In some embodiments, R⁸ is hydrogen or ethyl. In someembodiments, R⁸ is hydrogen.

In some embodiments, R⁹ and R¹⁰ are each independently hydrogen,—(C₁-C₆)-alkyl, —(C₃-C₈)-cycloalkyl, or C(O)R¹¹, wherein the—(C₁-C₆)-alkyl or —(C₃-C₈)-cycloalkyl are optionally substituted with—(C₁-C₆)-alkyl, —(C₃-C₈)-cycloalkyl, —NR¹¹R¹², —S¹¹, or heterocyclyl. Insome embodiments, R⁹ and R¹⁰ are each independently hydrogen,—(C₁-C₆)-alkyl, or —(C₃-C₈)-cycloalkyl. In some embodiments, R⁹ and R¹⁰are each independently hydrogen, —(C₁-C₆)-alkyl, or —(C₃-C₈)-cycloalkyl.In some embodiments, R⁹ and R¹⁰ are each independently hydrogen, or—(C₁-C₆)-alkyl. In some embodiments, R⁹ and R¹⁰ are independently—(C₁-C₃)-alkyl, or —(C₃-C₈)-cycloalkyl.

In some embodiments, R⁹ and R¹⁰ together with the nitrogen atom to whichthey are attached form a 3 to 8-membered heterocycle, wherein any one ofthe ring carbon atoms is optionally replaced with a heteroatom, andwherein the heterocycle is optionally substituted with —(C₁-C₆)-alkyl.

In some embodiments, R¹³ is hydrogen, —(C₁-C₆)-alkyl,—(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷, —C(O)N(R⁷)₂, or —S(O)₂R⁷. Insome embodiments, R¹³ is hydrogen, —(C₁-C₃)-alkyl, —(C₃-C₅)-cycloalkyl,—C(O)R⁷, —C(O)OR⁷, —C(O)N(R⁷)₂, or —S(O)₂R⁷. In some embodiments, R¹³ ishydrogen, —(C₁-C₃)-alkyl, or —(C₃-C₅)-cycloalkyl. In some embodiments,R¹³ is hydrogen, or —(C₃-C₅)-cycloalkyl. In some embodiments, R¹³ ishydrogen, or —(C₁-C₃)-alkyl. In some embodiments, R¹³ is hydrogen,—(C₁-C₃)-alkyl or cyclopropyl. In some embodiments, R¹³ is hydrogen or—(C₁-C₃)-alkyl. In some embodiments, R¹³ is hydrogen or cyclopropyl. Insome embodiments, R¹³ is hydrogen. In some embodiments, R¹³ is—(C₁-C₃)-alkyl or cyclopropyl. In some embodiments, R¹³ is hydrogen. Insome embodiments, R¹³ is —(C₁-C₃)-alkyl. In some embodiments, R¹³ isethyl. In some embodiments, R¹³ is cyclopropyl.

In some embodiments, m is 0, 1 or 2. In some embodiments, m is 0 or 1.In some embodiments, m is 1 or 2. In some embodiments, m is 0. In someembodiments, m is 1. In some embodiments, m is 2.

In some embodiments, n is 0, 1 or 2. In some embodiments, n is 0 or 1.In some embodiments, n is 1 or 2. In some embodiments, n is 0. In someembodiments, n is 1. In some embodiments, n is 2.

In some embodiments, m is 1 and n is 1. In some embodiments, m is 2 andn is 1. In some embodiments, m is 1 and n is 2. In some embodiments, mis 0 and n is 1. In some embodiments, m is 0 and n is 2.

In some embodiments, Y is NR⁵. In some embodiments, Y is O or S. In someembodiments, Y is NH.

In some embodiments, V is a bond, W is a bond, X is —(C₁-C₃)-alkyl; Y isNR⁵; and m and n are each 1 or 2.

In some embodiments, A is NR⁴, V is a bond or C(O), W is a bond or NR¹³,X is —(C₁-C₃)-alkyl; Y is NR⁵; R¹ is halogen or —(C₁-C₃)-haloalkyl; R²is —OR⁶; R³ is hydrogen, —OMe, —CF3, —CN or halogen; R⁵ is hydrogen,—(C₁-C₃)-alkyl, —(C₁-C₃)-cycloalkyl, or C(O)R⁷; R⁶ is —(C₁-C₃)-alkyl or—(C₁-C₃)-haloalkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen; R¹³ ishydrogen or —(C₁-C₃)-alkyl; m is 0 or 1; and n is 2, provided that thesum of m+n is an integer from 2-3.

In some embodiments, A is NR⁴, V is a bond or C(O), W is a bond or NR¹³,X is —(C₁-C₃)-alkyl; Y is NR⁵; R¹ is halogen; R² is —OR⁶; R³ ishydrogen, —OMe, —CF3, CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, orC(O)R⁷; R⁶ is —(C₁-C₃)-alkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen; R¹³is hydrogen; m is 0 or 1; and n is 2, provided that the sum of m+n is aninteger from 2-3.

In some embodiments, A is 0, V is a bond or C(O), W is a bond or NH, Xis —CH₂—; Y is NR⁵; R¹ is halogen; R² is OCH₃; R³ is hydrogen, —OMe,—CF3, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or C(O)CH₃; R⁸ ishydrogen; m is 0 or 1; and n is 2, provided that the sum of m+n is aninteger from 2-3.

In some embodiments, X is CH₂; Y is NR⁵; R¹ is hydrogen, halogen or—(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen, —OMe, —CF3, —CN orhalogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or —(C₃-C₅)-cycloalkyl; R⁶ ishydrogen, —(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl; and R⁸ is hydrogen,—(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl.

In some embodiments, V is a bond, W is a bond, X is CH₂; Y is NR⁵; R¹ ishydrogen, halogen or —(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen,—OMe, —CF3, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or—(C₃-C₅)-cycloalkyl; R⁶ is hydrogen, —(C₁-C₃)-alkyl, or—(C₁-C₃)-haloalkyl; and R⁸ is hydrogen, —(C₁-C₃)-alkyl, or—(C₁-C₃)-haloalkyl.

In some embodiments, X is CH₂; Y is NR⁵; R¹ is hydrogen, halogen or—(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen, —OMe, —CF3, —CN orhalogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or —(C₃-C₅)-cycloalkyl; R⁶ ishydrogen, —(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl; R⁶ is hydrogen,—(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl; R⁸ is hydrogen, —(C₁-C₃)-alkyl,or —(C₁-C₃)-haloalkyl; and m and n are each 1 or 2.

In some embodiments, V is a bond, W is a bond, X is CH₂; Y is NR⁵; R¹ ishydrogen, halogen or —(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen,—OMe, —CF3, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or—(C₃-C₅)-cycloalkyl; R⁶ is hydrogen, —(C₁-C₃)-alkyl, or—(C₁-C₃)-haloalkyl; R⁶ is hydrogen, —(C₁-C₃)-alkyl, or—(C₁-C₃)-haloalkyl; R⁸ is hydrogen, —(C₁-C₃)-alkyl, or—(C₁-C₃)-haloalkyl; and m and n are each 1 or 2.

In some embodiments, V is a bond, W is a bond, A is NH; X is CH₂; Y isNR⁵; R¹ is halogen or —(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen,—OMe, —CF3, —CN or halogen; R⁵ is hydrogen or —(C₁-C₃)-alkyl; and R⁶ ishydrogen, —(C₁-C₂)-alkyl, or —(C₁-C₂)-haloalkyl.

In some embodiments, A is NH; X is CH₂; Y is NR⁵; R¹ is halogen or—(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen, —OMe, —CF3, —CN orhalogen; R⁵ is hydrogen or —(C₁-C₃)-alkyl; and R⁶ is hydrogen,—(C₁-C₂)-alkyl, or —(C₁-C₂)-haloalkyl.

In some embodiments, A is NH; X is CH₂; Y is NR⁵; R¹ is halogen or—(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen, —OMe, —CF3, —CN orhalogen; R⁵ is hydrogen or —(C₁-C₃)-alkyl; R⁶ is hydrogen,—(C₁-C₂)-alkyl, or —(C₁-C₂)-haloalkyl; m is 1; and n is 2.

In some embodiments, A is NH; X is CH₂; Y is NR⁵; R¹ is halogen; R² is—OCH₃; R³ is hydrogen, —OMe, —CF3, —CN or halogen; R⁵ is hydrogen or—(C₁-C₃)-alkyl; m is 1; and n is 2.

In some embodiments, R¹ and R² are not both hydrogen when V and W areeach a bond, Y is NR⁵, A is NR⁴, X is CO, n=2 and m=1.

In some embodiments, A is NR⁴; V is a bond or C(O); W is a bond or NR¹³;X is —(C₁-C₃)-alkyl; Y is NR⁵; R¹ is halogen or —(C₁-C₃)-haloalkyl; R²is —OR⁶; R³ is hydrogen, —OMe, —CF3, —CN or halogen; R⁵ is hydrogen,—(C₁-C₃)-alkyl, —(C₁-C₃)-cycloalkyl, or —C(O)R⁷; R⁶ is —(C₁-C₃)-alkyl or—(C₁-C₃)-haloalkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen or—(C₁-C₃)-alkyl; R¹³ is hydrogen or —(C₁-C₃)-alkyl; and m and n areindependently 0, 1 or 2, provided that the sum of m+n is an integer from2-3.

In some embodiments, A is NH; V is a bond or C(O); W is a bond or NR¹³;X is —(C₁-C₃)-alkyl; Y is NR⁵; R¹ is halogen; R² is —OR⁶; R³ ishydrogen, —OMe, —CF3, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or—C(O)R⁷; R⁶ is —(C1-C₃)-alkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen or—(C₁-C₃)-alkyl; R¹³ is hydrogen; m is 0 or 1; and n is 2.

In some embodiments, A is NH; V is a bond or C(O); W is a bond or NH; Xis —CH₂—; Y is NR⁵; R¹ is halogen; R² is —OCH₃; R³ is hydrogen, —OMe,—CF3, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or —C(O)CH₃; R⁸ ishydrogen or —(C₁-C₃)-alkyl; m is 0 or 1; and n is 2.

In some embodiments, V and W are each a bond, or V is C(O) and W isNR¹³; X is —(C₁-C₃)-alkyl; Y is NR⁵; and m and n are each 0, 1 or 2,provided that the sum of m+n is an integer from 2-3.

In some embodiments, A is NH; V is C(O); W is NH; X is CH₂; Y is NR⁵; R¹is halogen or —(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen, —OMe,—CF3, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or C(O)R⁷; R⁶ ishydrogen, —(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl; R⁷ is hydrogen or—(C₁-C₃)-alkyl; R⁸ is hydrogen, —(C₁-C₃)-alkyl, —(C₁-C₃)-haloalkyl,—(C₃-C₅)-cycloalkyl, —NR⁹R¹⁰; m is 0 or 1; and n is 2.

In some embodiments, A is NH; V is C(O); W is NH; X is CH₂; Y is NR⁵; R¹is halogen; R² is —OCH₃; R³ is hydrogen, —OMe, —CF3, —CN or halogen; R⁵is hydrogen, —(C₁-C₃)-alkyl, or C(O)R⁷; R⁷ is —(C₁-C₃)-alkyl; R⁸ ishydrogen or —(C₁-C₃)-alkyl; m is 0; and n is 2.

In some embodiments, A is NH; V and W are each a bond, or V is C(O) andW is NH; X is CH₂; Y is NR⁵; R¹ is halogen or —(C₁-C₃)-haloalkyl; R² is—OR⁶; R³ is hydrogen, —OMe, —CF3, —CN or halogen; R⁵ is hydrogen,—(C₁-C₃)-alkyl, or C(O)R⁷; R⁶ is hydrogen, —(C₁-C₂)-alkyl, or—(C₁-C₂)-haloalkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen,—(C₁-C₃)-alkyl, —(C₁-C₃)-haloalkyl, or —(C₃-C₅)-cycloalkyl; m is 0 or 1;and n is 2.

In some embodiments, A is NH; V and W are each a bond; X is CH₂; Y isNR⁵; R¹ is chlorine; R² is —OCH₃; R³ is —CN; R⁵ is hydrogen or—(C₁-C₃)-alkyl; R⁸ is hydrogen or —(C₁-C₃)-alkyl; m is 1; and n is 2.

In some embodiments, A is NH; V and W are each a bond; X is CH₂; Y isNC(O)CH₃; R¹ is halogen; R² is —OCH₃; R³ is hydrogen, —OMe, —CF3, —CN orhalogen; R⁸ is hydrogen; m is 1; and n is 2.

In another aspect, the invention is directed to compositions comprisinga compound of formula (I), (Ia), (Ib), or (Ic) and a pharmaceuticallyacceptable carrier.

In another aspect, the invention is directed to a method of inhibitingphosphodiesterase comprising contacting a phosphodiesterase with acompound of formula (I), (Ia), (Ib), or (Ic) or a composition comprisinga compound of formula (I), (Ia), (Ib), or (Ic). In some embodiments, thephosphodiesterase is PDE5.

In another aspect, the invention is directed to a method of treatingneurodegenerative disease in a subject comprising administration of atherapeutically effective amount of a compound of formula (I), (Ia),(Ib), or (Ic). In some embodiments, the disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of treatingneurodegenerative disease in a subject comprising administration of atherapeutically effective amount of a composition comprising a compoundof formula (I), (Ia), (Ib), or (Ic). In some embodiments, the disease isAlzheimer's Disease.

In another aspect, the invention is directed to a method of increasinglong-term potentiation in a subject comprising administration of atherapeutically effective amount of a compound of formula (I), (Ia),(Ib), or (Ic). In some embodiments, the subject has a neurodegenerativedisease. In some embodiments, the neurodegenerative disease isAlzheimer's Disease.

In another aspect, the invention is directed to a method of increasinglong-term potentiation in a subject comprising administration of atherapeutically effective amount of a composition comprising a compoundof formula (I), (Ia), (Ib), or (Ic). In some embodiments, the subjecthas a neurodegenerative disease. In some embodiments, theneurodegenerative disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingmemory in a subject comprising administration of a therapeuticallyeffective amount of a compound of formula (I), (Ia), (Ib), or (Ic). Insome embodiments, the subject has a neurodegenerative disease. In someembodiments, the neurodegenerative disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingmemory in a subject comprising administration of a therapeuticallyeffective amount of a composition comprising a compound of formula (I),(Ia), (Ib), or (Ic). In some embodiments, the subject has aneurodegenerative disease. In some embodiments, the neurodegenerativedisease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject comprising administration of atherapeutically effective amount of a compound of formula (I), (Ia),(Ib), or (Ic). In some embodiments, synaptic function comprises synapticplasticity. In some embodiments, synaptic plasticity comprises learning,memory, or a combination thereof. In some embodiments, synapticplasticity comprises long term potentiation (LTP). In some embodiments,the subject has a neurodegenerative disease. In some embodiments, theneurodegenerative disease is Alzheimer's Disease.

In another aspect, the invention is directed to a method of improvingsynaptic function in a subject comprising administration of atherapeutically effective amount of a composition comprising a compoundof formula (I), (Ia), (Ib), or (Ic). In some embodiments, synapticfunction comprises synaptic plasticity. In some embodiments, synapticplasticity comprises learning, memory, or a combination thereof. In someembodiments, synaptic plasticity comprises long term potentiation (LTP).In some embodiments, the subject has a neurodegenerative disease. Insome embodiments, the neurodegenerative disease is Alzheimer's Disease.

Another aspect of the invention provides a method for increasing memoryretention in a subject afflicted with a neurodegenerative disease, themethod comprising administering to a subject a therapeutic amount of acompound of formula (I), (Ia), (Ib), or (Ic) or a composition comprisinga compound of formula (I), (Ia), (Ib), or (Ic).

In some embodiments, a compound of formula (I), (Ia), (Ib), or (Ic) isadministered. In some embodiments, a composition comprising a compoundof formula (I), (Ia), (Ib), or (Ic) is administered.

Exemplary neurodegenerative diseases and methods of treatment thereforare also described in WO 2010/074783, WO2011/072243, and WO2012/088420,each herein incorporated by reference in its entirety.

Compounds of formula (I), (Ia), (Ib), or (Ic) can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions can comprise a compound of formula (I), (Ia), (Ib), or (Ic)and a pharmaceutically acceptable carrier. Thus, in some embodiments,the compounds of the invention are present in a pharmaceuticalcomposition.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

Any of the therapeutic applications described herein can be applied toany subject in need of such therapy, including, for example, a mammalsuch as a mouse, a rat, a dog, a cat, a cow, a horse, a rabbit, amonkey, a pig, a sheep, a goat, or a human. In some embodiments, thesubject is a mouse, rat or human. In some embodiments, the subject is amouse. In some embodiments, the subject is a rat. In some embodiments,the subject is a human.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyetheylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, itcan be useful to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the PDE5inhibitor compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated herein, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated herein. In the case of sterile powders for thepreparation of sterile injectable solutions, examples of usefulpreparation methods are vacuum drying and freeze-drying which yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

Benzonaphthyridine derivatives are synthesized by methods within thepurview of the ordinarily skilled artisan. Exemplary methods by whichbenzonaphthyridine derivatives can be synthesized are as follows.

Method A: Benzonaphthyridine derivatives can be synthesized, forexample, starting from 2-aminobenzoic acid derivative (Scheme 1).

A mixture of 2-aminobenzoic acid derivative and cyclic amino ketonederivative (optionally containing an alkyl group or other protectinggroup at the nitrogen atom) can be heated in, for example, POCl₃ toobtain a benzonaphthyridine. Re-installation of the protecting group (ifit is removed during benzonaphthyridine formation) can be achieved byknown methods such as those described in, for example, Protecting Groupsin Organic Synthesis, 4^(th) Edition, by Peter Wuts & Theodora Greene(Wiley 2006), hereby incorporated by reference in its entirety.Subsequent treatment with a phenylalkylamine and a salt such as, forexample, a sodium halide, in a solvent such as a phenol can be heated toobtain the elaborated benzonphthyridine derivative. Optional protectionof the nitrogen atom of the phenylalkylamine can be achieved by knownmethods, such as those described in, for example, Protecting Groups inOrganic Synthesis, 4^(th) Edition, by Peter Wuts & Theodora Greene(Wiley 2006), hereby incorporated by reference in its entirety. Theskilled artisan will recognize selection of an appropriate protectinggroup based on desired properties and orthogonality with otherprotecting groups at, for example, the piperidine nitrogen atom.Optional deprotection of the piperidine nitrogen can be achieved byknown methods, such as those described in, for example, ProtectingGroups in Organic Synthesis, 4^(th) Edition, by Peter Wuts & TheodoraGreene (Wiley 2006) (hereby incorporated by reference in its entirety),followed by incorporation of the desired R⁵ group via reductiveamination of an R⁵—CHO in the presence of a reducing agent such as aborohydride or via alkylation of R⁵-LG (wherein LG is a leaving groupsuch as, for example, halogen, sulfonate, etc.). Removal of protectinggroups can be achieved by known methods, such as those described in, forexample, Protecting Groups in Organic Synthesis, 4^(th) Edition, byPeter Wuts & Theodora Greene (Wiley 2006) (hereby incorporated byreference in its entirety).

Method B: Benzonaphthyridine derivatives can be synthesized, forexample, starting from 2-aminobenzoic acid derivative (Scheme 2).

An aniline derivative in a solvent such as water can be treated withchloral hydrate, acid (such as HCl), a group I sulfate (such as sodiumsulfate), hydroxylamine and heated to generate a hydroxyl-imine. Thehydroxyl-imine can be treated with an acid (such as sulfuric acid) andheated to generate an indoline-2,3-dione. Subsequent treatment with anoxidant such as hydrogen peroxide in a base such as sodium hydroxide cangenerate a 2-aminobenzoic acid. Optional conversion of R³ to —CN can beachieved via treatment with copper cyanide in a solvent such as NMPprior to further processing.

The 2-aminobenzoic acid derivative and cyclic amino ketone derivative(optionally containing an alkyl group or other protecting group at thenitrogen atom) can be heated in POCl₃ to obtain a benzonaphthyridine.Re-installation of the protecting group (if it is removed duringbenzonaphthyridine formation) can be achieved by known methods such asthose described in, for example, Protecting Groups in Organic Synthesis,4^(th) Edition, by Peter Wuts & Theodora Greene (Wiley 2006), herebyincorporated by reference in its entirety. Subsequent treatment with aphenylalkylamine and a salt such as, for example, a sodium halide, in asolvent such as a phenol can be heated to obtain the elaboratedbenzonphthyridine derivative. Optional protection of the nitrogen atomof the phenylalkylamine can be achieved by known methods, such as thosedescribed in, for example, Protecting Groups in Organic Synthesis,4^(th) Edition, by Peter Wuts & Theodora Greene (Wiley 2006), herebyincorporated by reference in its entirety. The skilled artisan willrecognize selection of an appropriate protecting group based on desiredproperties and orthogonality with other protecting groups at, forexample, the piperidine nitrogen atom. Optional deprotection of thepiperidine nitrogen can be achieved by known methods, such as thosedescribed in, for example, Protecting Groups in Organic Synthesis,4^(th) Edition, by Peter Wuts & Theodora Greene (Wiley 2006) (herebyincorporated by reference in its entirety), followed by incorporation ofthe desired R⁵ group via reductive amination of an R⁵—CHO in thepresence of a reducing agent such as a borohydride or via alkylation ofR⁵-LG (wherein LG is a leaving group such as, for example, halogen,sulfonate, etc.). Removal of protecting groups can be achieved by knownmethods, such as those described in, for example, Protecting Groups inOrganic Synthesis, 4^(th) Edition, by Peter Wuts & Theodora Greene(Wiley 2006) (hereby incorporated by reference in its entirety).

Method C: Benzonaphthyridine derivatives can also be synthesized, forexample, starting from 2-aminobenzonitrile derivative (Scheme 3).

A mixture of 2-aminobenzonitrile derivative and cyclic amino ketonederivative can be heated in the presence of a strong acid. Strong acidsemployed in this transformation can be any organic or inorganic acid,such as, for example, polyphosphoric, trifluoroacetic, acetic, andsulfonic acid. Subsequent treatment with a phenylalkyl moiety containinga leaving group (LG) and a base such as, for example, a trialkylamine,can form the elaborated benzonphthyridine derivative.

In addition to the aforementioned general methods, other methods such asthose described in, for example, U.S. Pat. Nos. 3,674,790 and 6,294,547(each herein incorporated by reference in its entirety) can also be usedto obtain the benzonaphthyridine derivatives. The ordinarily skilledartisan will recognize variations of the methods described herein and ofthe methods described in the references herein cited to synthesize otherbenzonaphthyridine derivatives within the scope of the invention.

A PDE5 inhibitor can decrease the activity of a PDE5 molecule in vivoand/or in vitro. In one embodiment, a PDE5 inhibitor can decrease PDE5activity by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%, or 100%.

In some embodiments, the compounds of the invention exhibit inhibitionof PDE5 with an IC50 less than about 1 μM. In some embodiments, thecompounds of the invention exhibit inhibition of PDE5 with an IC50 lessthan about 500 nM. In some embodiments, the IC50 is less than about 250nM, less than about 100 nM, less than about 50 nM, less than about 25nM, less than about 10 nM, less than about 5 nM, or less than about 1nM.

In some embodiments, the compounds of formula (I), (Ia), (Ib), or (Ic)are selective inhibitors of PDE5. In some embodiments, the compoundsexhibit inhibition of PDE5 at lower concentrations than they inhibitother PDE subtypes. In some embodiments, other PDE subtypes may includeany of PDE1-PDE4, and PDE6-PDE11 or any combination thereof. In someembodiments, the other PDE subtype is PDE1. In some embodiments, theother PDE subtype is PDE6. In some embodiments, the other PDE subtype isPDE9.

It will recognized that one or more features of any embodimentsdisclosed herein may be combined and/or rearranged within the scope ofthe invention to produce further embodiments that are also within thescope of the invention. Those skilled in the art will recognize, or beable to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be within the scope of thepresent invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

The invention is further described by the following non-limitingExamples.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 18-bromo-N-(3-chloro-4-methoxybenzyl)-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridin-10-amine

8-bromo-10-chloro-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine,2

A mixture of 1 (9.25 mmol) and 1-methylpiperidin-4-one (9.25 mmol) inPOCl₃ (10 mL) was heated at 60° C. for 6 h. The excess POCl₃ wasevaporated off; the residue was treated with iced H₂O and NaHCO₃ andextracted with AcOEt (3×50 mL). The organic layer was dried over Na₂SO₄,filtered and evaporated under reduced pressure. The desired compound waspurified by triturating with Et₂O (42% of yield). ¹H NMR (300 MHz,CDCl₃) δ 8.33 (d, 1H, J=2.1 Hz), 7.86 (d, 1H, J=9.0 Hz), 7.77 (dd, 1H,J₁=2.1, J₂=9.0 Hz), 3.99 (s, 2H), 3.34 (t, 2H, J=5.7 Hz), 3.03 (t, 2H,J=5.7 Hz), 2.69 (s, 3H).

A mixture of 2 (1.92 mmol), 3-chloro-4-methoxybenzylamine hydrochloride(2.11 mmol), NaI (0.1 mmol), and phenol (3.84 mmol) was heated at 130°C. for 1.5 h. After cooling the reaction down, Et₂O (20 mL) was addedand washed with 1N NaOH (3×20 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated under reduced pressure. The residue waspurified by Flash Chromatography (AcOEt: MeOH 8:2) to give the desiredcompound A (38% yield). ¹H NMR (300 MHz, CDCl₃) δ 8.04 (d, 1H, J=2.1Hz), 7.80 (d, 1H, J=8.7 Hz), 7.64 (dd, 1H, J₁=2.1, J₂=9.0 Hz), 7.38 (d,1H, J=2.1 Hz), 7.16 (dd, 1H, J₁=2.1, J₂=8.4 Hz), 6.91 (d, 1H, J=8.4 Hz),4.47 (d, 2H, J=5.7 Hz), 3.91 (s, 3H), 3.54 (s, 2H), 3.18 (t, 2H, J=6.3Hz), 2.81 (t, 2H, J=6.3 Hz), 2.50 (s, 3H).

Example 210-[(3-chloro-4-methoxybenzyl)amino]-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

2-amino-5-cyanobenzoic acid, 3

2-amino-5-bromobenzoic acid (2.31 mmol) and CuCN (2.78 mmol) were heatedto reflux in NMP (5 mL) for 3 h. The reaction mixture was poured into asolution of FeCl₃ 3H₂O (3.0 g) in H₂O (3 mL) and HCl (0.5 mL) andstirred at 60° C. for 1 h. After cooling the reaction down, Et₂O (100mL) was added and the two phases were separated. The organic layer waswashed with HCl 1N (50 mL) and H₂O (2×50 mL), dried over Na₂SO₄,filtered and evaporated under reduced pressure. 2-amino-5-cyanobenzoicacid (3) was obtained (57% of yield) by triturating from CH₂Cl₂.

¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, 1H, J=1.8 Hz), 7.71 (dd, 1H, J₁=1.8,J₂=8.7 Hz), 7.32 (br s, 1H), 6.75 (d, 1H, J=8.7 Hz).

10-chloro-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,4

A mixture of 1 (1.23 mmol) and 1-methylpiperidin-4-one (1.23 mmol) inPOCl₃ (2 mL) was heated at 60° C. for 6 h. The excess POCl₃ wasevaporated off; the residue was treated with iced H₂O and NaHCO₃ andextracted with CH₂Cl₂ (3×25 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated under reduced pressure. Purification byFlash Chromatography (5% MeOH in AcOEt) gave the desired compound (55%of yield). 1H NMR (300 MHz, CDCl₃) δ 8.58 (s, 1H), 8.07 (d, 1H, J=8.7Hz), 7.83 (d, 1H, J=8.7 Hz), 3.85 (s, 2H), 3.30 (t, 2H, J=6.0 Hz), 2.89(t, 2H, J=6.0 Hz), 2.60 (s, 3H).

10-chloro-2-ethyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,5

A mixture of 1 (1.23 mmol) and 1-ethylpiperidin-4-one (1.23 mmol) inPOCl₃ (2 mL) was heated at 60° C. for 6 h. The excess POCl₃ wasevaporated off; the residue was treated with iced H₂O and NaHCO₃ andextracted with CH₂Cl₂ (3×25 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated under reduced pressure. Purification byFlash Chromatography (5% MeOH in CH₂Cl₂) gave the desired compound (26%of yield). ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, 1H, J=1.8 Hz), 8.06 (d,1H, J=8.7 Hz), 7.82 (dd, 1H, J₁=1.8, J₂=8.4 Hz), 3.91 (s, 2H), 3.30 (t,2H, J=5.7 Hz), 2.93 (t, 2H, J=5.7 Hz), 2.75 (q, 2H, J=7.2 Hz), 1.26 (t,3H, J=7.2 Hz).

10-[(3-chloro-4-methoxybenzyl)amino]-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

A mixture of 4 (0.12 mmol), 3-chloro-4-methoxybenzylamine hydrochloride(0.12 mmol), NaI (0.006 mmol), and phenol (0.12 mmol) was heated at 130°C. for 2.5 h. The reaction mixture was diluted with Et₂O (10 mL) andwashed with 1N NaOH (3×5 mL). The organic layer was dried over Na₂SO₄,filtered and evaporated under reduced pressure. The residue was purifiedby Flash Chromatography (AcOEt: MeOH 8:2) to give the desired compound B(33% of yield). MS ESI (m/z) 394 (M+H)⁺, ¹H NMR (300 MHz, CDCl₃) δ 8.32(d, 1H, J=1.2 Hz), 7.97 (d, 1H, J=8.7 Hz), 7.71 (dd, 1H, J₁=1.8, J₂=8.7Hz), 7.37 (d, 1H, J=2.1 Hz), 7.18 (dd, 1H, J₁=2.4, J₂=8.7 Hz), 6.94 (d,1H, J=8.4 Hz), 4.57 (d, 2H, J=6.0 Hz), 4.11 (br s, 1H), 3.92 (s, 3H),3.54 (s, 2H), 3.22 (t, 2H, J=6.3 Hz), 2.83 (t, 2H, J=6.3 Hz), 2.52 (s,3H).

Example 310-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

A mixture of 5 (0.18 mmol), 3-chloro-4-methoxybenzylamine hydrochloride(0.18 mmol), NaI (0.009 mmol), and phenol (0.18 mmol) was heated at 130°C. for 2.5 h (Scheme 4). The reaction mixture was diluted with Et₂O (30mL) and washed with 1N NaOH (3×1 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated under reduced pressure. The residue waspurified by Flash Chromatography (5% MeOH in AcOEt) to give the desiredcompound C (38% of yield). MS ESI (m/z) 407 (M+H), 1H NMR (300 MHz,CDCl₃) δ 8.32 (d, 1H, J=1.2 Hz), 7.97 (d, 1H, J=8.7 Hz), 7.71 (dd, 1H,J₁=1.8, J₂=8.7 Hz), 7.37 (d, 1H, J=2.1 Hz), 7.17 (dd, 1H, J₁=2.1, J₂=8.4Hz), 6.94 (d, 1H, J=8.4 Hz), 4.56 (d, 2H, J=5.7 Hz), 4.12 (br s, 1H),3.92 (s, 3H), 3.59 (s, 2H), 3.22 (t, 2H, J=6.0 Hz), 2.87 (t, 2H, J=6.0Hz), 2.66 (q, 2H, J=7.2 Hz), 1.18 (t, 3H, J=7.2 Hz).

Example 410-[(3-chloro-4-methoxybenzyl)amino]-6-ethyl-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

N-(4-bromo-2-ethylphenyl)-2-(hydroxyimino)acetamide, 7

To a suspension of 4-bromo-2-ethylaniline (6) (5.0 mmol) in 50 mL of H₂Owas added HCl cone. (0.5 mL), 4.4 g of Na₂SO₄, and NH₂OH hydrochloride(14.9 mmol), followed by addition of chloral hydrate (5.5 mmol). Thereaction mixture was heated to 90° C. for 1 h. After cooling down toroom temperature, the aqueous phase was extracted with AcOEt (3×50 mL).The organic layers were dried over Na₂SO₄, filtered and evaporated togive compound 7. ¹H NMR (300 MHz, CDCl₃) δ 8.22 (br s, 1H), 7.93-7.90(m, 2H), 7.60 (s, 1H), 7.37-7.34 (m, 2H), 2.60 (q, 2H, J=7.5 Hz), 1.25(t, 3H, J=7.2 Hz).

5-bromo-7-ethylindoline-2,3-dione, 8

To a solution of sulfuric acid (10 mL) and H₂O (1 mL) at 80° C. wasadded 7 (3.68 mmol) in small portions over 20 minutes. The reactionmixture was stirred for 15 min at 80° C. After cooling the reactiondown, 20 mL of iced-water was added and the mixture was extracted withAcOEt (3×50 mL). The organic layers were dried over Na₂SO₄, filtered andevaporated to give the desired product. ¹H NMR (300 MHz, CDCl₃) δ 8.85(s, 1H), 7.58 (d, 1H, J=1.5 Hz), 7.58-7.54 (m, 1H), 2.60 (q, 2H, J=7.5Hz), 1.29 (t, 3H, J=7.5 Hz).

2-amino-5-bromo-3-ethylbenzoic acid, 9

To a suspension of 8 (1.38 mmol) in 10 mL of NaOH 10% a solution of H₂O₂(0.5 mL) in 4.5 mL of H₂O was added dropwise. The mixture was stirredovernight at room temperature. The reaction mixture was filtered and thefiltrate was acidified by adding HCl conc., the resulting precipitate(9) was filtered and dried. ¹H NMR (300 MHz, CDCl₃) δ 7.95 (d, 1H, J=2.1Hz), 7.32 (d, 1H, J=2.4 Hz), 2.49 (q, 2H, J=7.2 Hz), 1.28 (t, 3H J=7.2Hz).

2-amino-5-cyano-3-ethylbenzoic acid, 10

A mixture of 9 (3.3 mmol) and CuCN (3.9 mmol) was reflux in 3 mL of NMPfor 4 h. The reaction mixture was poured into a warm solution of NaCN(33% w/v) and vigorously shaken. After cooling down, the reaction wasextracted with AcOEt (50 mL) and the organic phase was discarded. Theaqueous layer was acidified by adding HCl conc. and the resultingprecipitate 10 was collected by filtration. 1H NMR (300 MHz, CDCl₃) δ8.18 (d, 1H, J=1.8 Hz), 7.26 (s, 1H), 6.49 (br s, 1H), 2.51 (q, 2H,J=7.2 Hz), 1.30 (t, 3H, J=7.2 Hz).

10-chloro-6-ethyl-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,11

A mixture of 10 (0.64 mmol) and 1-methylpiperidin-4-one (0.64 mmol) inPOCl₃ (2 mL) was heated at 60° C. for 6 h. The excess POCl₃ wasevaporated off; the residue was treated with iced H₂O and NaHCO₃ andextracted with CH₂Cl₂ (3×10 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated under reduced pressure. The desiredcompound was purified by triturating with Et₂O. ¹H NMR (300 MHz, CDCl₃)δ 8.42 (d, 1H, J=1.8 Hz), 7.65 (m, 1H), 3.82 (s, 2H), 3.31-3.23 (m, 4H),2.87 (t, 2H J=5.7 Hz), 2.58 (s, 3H), 1.34 (t, 3H J=7.2 Hz).

10-[(3-chloro-4-methoxybenzyl)amino]-6-ethyl-2-methyl-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

A mixture of 11 (0.18 mmol), 3-chloro-4-methoxybenzylamine hydrochloride(0.18 mmol), NaI (0.009 mmol), and phenol (0.18 mmol) was heated at 130°C. for 4 h. The reaction mixture was diluted with Et₂O (30 mL) andwashed with 1N NaOH (3×10 mL). The organic layer was dried over Na₂SO₄,filtered and evaporated under reduced pressure. The final product D wasobtained by Flash Chromatography (AcOEt: MeOH 9:1). MS ESI (m/z) 421(M+H)⁺, ¹H NMR (300 MHz, CDCl₃) δ 8.16 (d, 1H, J⁼1.5 Hz), 7.56 (s, 1H),7.36 (d, 1H, J=2.1 Hz), 7.18-7.16 (m, 1H), 6.93 (d, 1H, J=8.4 Hz), 4.48(s, 2H), 3.92 (s, 3H), 3.54 (s, 2H), 3.25-3.21 (m, 4H), 2.89 (t, 2H,J=6.0 Hz), 2.51 (s, 3H), 1.35 (t, 3H, J=7.5 Hz).

Example 56-[(3-chloro-4-methoxybenzyl)amino]-5-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[5,6-b]quinoline-8-carbonitrile

Methyl 2,4-dichloro-6-cyanoquinoline-3-carboxylate, 13

To a solution of methyl 2-amino-5-cyanobenzoate (5.68 mmol) in DCM (10mL) was added triethyamine (8.52 mmol) followed by methyl3-chloro-3-oxopropionate (7.38 mmol). The resulting reaction mixture wasstirred at ambient temperature overnight. The reaction mixture waspartitioned between EtOAc and 1M HCl and the layers then separated. Theorganic layer washed sequentially with saturated aqueous NaHCO₃ followedby brine. The organic layer was dried over Na₂SO₄, filtered, andconcentrated in vacuo. The resulting residue was carried on withoutfurther purification.

The residue from above was treated with 0.5M NaOMe in MeOH (6.83 mmol).The heterogenous mixture was stirred at ambient temperature for 30 min.Diethyl ether was then added to the reaction mixture and the solid wascollected via vacuum filtration, washing with ether. The obtained solidwas carried on without further purification.

To a flask containing the solid from above cooled to 0° C. was addedPOCl₃ (15 mL). The mixture became warm and bubbled. The resultingreaction mixture was heated to 90° C. for 3 h. Cooled the dark reactionmixture to ambient temperature and poured it very slowly into stirringsaturated aqueous NaHCO₃ cooled to 0° C. Solid NaHCO₃ was then addeduntil the solution was basic. The aqueous solution was extracted withEtOAc (3×). The combined organic layers were dried over Na₂SO₄,filtered, and concentrated in vacuo. The obtained residue was purifiedvia flash column chromatography, eluting with 9:1/hexanes:EtOAc, toyield the intermediate 13 (29% over 3 steps). MS ESI (nm/z) 281 (M+H)⁺;¹H NMR (300 MHz, CDCl₃) δ 8.63 (d, 1H, J==1.8 Hz), 8.16 (d, 1H, J=8.4Hz), 8.00 (dd, 1H, J₁=1.8, J₂=8.4 Hz) 4.08 (s, 3H); ¹³C NMR (CDCl₃, 75MHz) (163.4, 148.9, 148.1, 141.8, 133.3, 130.8, 130.6, 128.8, 124.3,117.7, 112.8, 54.1.

Methyl2-chloro-4-[(3-chloro-4-methoxybenzyl)amino]-6-cyanoquinoline-3-carboxylate,14

To a heterogenous mixture of quinoline 13 (3.56 mmol) and3-chloro-4-methoxy benzylamine.HCl (3.92 mmol) in NMP (15 mL) was added^(i)Pr₂NEt (8.90 mmol). The reaction mixture was heated to 80° C. for 3h. Subsequently, cooled the reaction mixture to ambient temperature andadded 1M aqueous HCl and EtOAc. The resulting layers were separated andthe organic layer washed with water (3×) followed by brine (lx). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue was dissolved in a minimum amount ofhot EtOAc and allowed to cool to ambient temperature. Hexanes was thenadded to the solution and the intermediate 14 (80% yield) precipitatedout and was collected by vacuum filtration. ¹H NMR (300 MHz, CDCl₃) δ8.20 (s, 1H), 7.95 (d, 1H, J=8.4 Hz), 7.82 (d, 1H, J=7.5 Hz), 7.38 (s,1H), 7.27-7.22 (m, 1H), 6.98 (d, 1H, J=8.4 Hz), 6.26 (s, 1H), 4.54 (d,2H J=4.8 Hz), 3.95 (s, 3H), 3.93 (s, 3H).

2-chloro-4[(3-chloro-4-ethoxybenzyl)amino]-6-cyanoquinoline-3-carboxylicacid, 15

To a solution of ester 14 (2.20 mmol) in 1,4-dioxane (70 mL) was addedLiOH (4.40 mmol) and water (20 mL). The resulting solution was stirredat ambient temperature overnight. The reaction mixture was partitionedbetween ether and 1M aqueous NaOH, the layers separated and the aqueouslayer acidified to a pH=2 using concentrated HCl. The precipitated solidwas collected via vacuum filtration to yield acid 15 (79% yield) thatwas used without further purification. ¹H NMR (300 MHz, DMSO) δ 9.02 (s,1H), 8.04-8.01 (m, 2H), 7.86 (d, 1H, J=8.7 Hz), 7.44 (s, 1H), 7.28 (d,1H, J=8.4 Hz), 7.12 (d, 1H, J=8.4; H), 4.57 (d, 2H, J=5.7 Hz), 3.84 (s,3H).

Perfluorophenyl2-chloro-4-[(3-chloro-4-methoxybenzyl)amino]-6-cyanoquinoline-3-carboxylate,16

To a solution of acid 15 (0.833 mmol) and pentafluorophenol (1.67 mmol)in DMF (5.6 mL) and 1,4-dioxane (2.2 mL), was added DCC (1.25 mmol). Thereaction mixture was stirred at ambient temperature overnight. Quenchedthe reaction mixture by addition of 1M aqueous HCl and extracted theaqueous layer with EtOAc (2×). The combined organic layers were washedwith 1M NaOH and brine. The combined organic layers were dried overNa₂SO₄, filtered, and concentrated in vacuo. The obtained residue waspurified via flash chromatography, eluting with 4:1/hexanes:EtOAc, togive the ester 16 (56% yield). ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, 1H,J=1.2 Hz), 7.99 (d, 1H, J=8.7 Hz), 7.89 (dd, 1H, J₁=1.2, J₂=8.7 Hz),7.40 (d, 1H, J=2.4 Hz), 7.30-7.24 (m, 1H), 7.01-6.98 (m, 2H), 4.69 (d,2H, J=4.8 Hz), 3.94 (s, 3H).

N-(2-aminoethyl)-2-chloro-4-[(3-chloro-4-methoxybenzyl)amino]-6-cyanoquinoline-3-carboxamide,17

To a solution of ester 16 (77.6 μmol) in THF (1.5 mL) was addedtriethyamine (0.116 mmol) followed by ethylenediamine (0.10 mmol). Theresulting reaction mixture was stirred at ambient temperature overnight.The reaction mixture was partitioned between EtOAc and saturated aqueousNaHCO₃, the layers separated, and the organic layer washed with 1M HCl(1×). The acidic aqueous layer was basicified to a pH=10 by addition ofsolid NaHCO₃ and then extracted with EtOAc (2×). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated in vacuo toyield 0.012 g (35% yield) of amine 17 that was used without furtherpurification. MS ESI (m/z) 444 (M+H)⁺; ¹H NMR (300 MHz, DMSO) δ 8.92 (s,1H), 8.52 (t, 1H, J=5.1 Hz), 7.93 (d, 1H, J=8.7 Hz), 7.81 (s, 1H), 7.76(d, 1H, J=8.7 Hz), 7.33 (s, 1H), 7.19 (d, 1H, J=8.4 Hz), 7.03 (d, 1H,J=8.4 Hz), 4.52 (s, 2H), 3.75 (s, 3H), 3.10-3.01 (m, 2H), 2.54 (t, 2H,J=6.3 Hz).

6-[(3-chloro-4-methoxybenzyl)amino]-5-oxo-2,3,4,5-tetrahydro1H-[1,4]diazepino[5,6-b]quinoline-8-carbonitrile, E

To a solution of amine 17 (27.0 μmol) in NMP (1.0 mL) was added TEA(40.5 μmol). The reaction mixture was heated to 100° C. overnight.Cooled to ambient temperature and partitioned between EtOAc and 1M HCl.The layers were separated and the aqueous layer was basicified to apH=10 by addition of solid NaHCO₃ and then extracted with EtOAc (2×).The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated in vacuo. The obtained residue was purified via flashchromatography, eluting with 19:1/CH₂Cl₂:MeOH, to yield the desiredproduct (18% yield). MS ESI (m/z) 408 (M+H)⁺; ¹H NMR (300 MHz, DMSO) δ8.72 (s, 1H), 8.28 (s, 1H), 7.78 (s, 1H), 7.68 (dd, 1H, J₁=1.5, J₂=8.4Hz), 7.34 (d, 1H, J=9), 7.31 (d, 1H, J=2.1 Hz), 7.19 (dd, 1H, J₁=2.1,J₂=8.4 Hz), 7.07 (d, 1H, J=8.4 Hz), 6.55 (s, 1H), 4.32 (d, 2H, J=5.7Hz), 3.81 (s, 3H), 3.1-3.05 (m, 4H).

Example 68-bromo-N-(3-chloro-4-methoxybenzyl)-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridin-10-amine

2-benzyl-8-bromo-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine,18

A mixture of 1 (13.9 mmol) and 1-benzylpiperidin-4-one (13.9 mmol) inPOCl₃ (50 mL) was heated at 60° C. for 6 h. The excess of POCl₃ wasevaporated off; the residue was treated with iced H₂O and NaOH 10% (50mL) and extracted with CH₂Cl₂ (3×50 mL). The organic layers were driedover Na₂SO₄, filtered and evaporated under reduced pressure. The desiredcompound (60% yield) was purified by flash chromatography (5% MeOH inDCM). MS ESI (m/z) 387 (M+H)⁺. ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, 1H,J=1.8 Hz), 7.86 (d, 1H, J=9.0 Hz), 7.75 (dd, 1H, J₁=2.1, J₂=8.7 Hz),7.43-7.31 (m, 5H), 3.92 (s, 2H), 3.82 (s, 2H), 3.21 (t, 2H, J=6.0 Hz),2.90 (t, 2H, J=6.0 Hz).

8-bromo-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine, 19

1-chloroethyl chloroformate (0.39 mmol) was added dropwise to a solutionof 18 (0.26 mmol) in DCE (2 mL) at 0° C. The mixture was heated toreflux for 2 h. DCE was evaporated off; the residue was dissolved inMeOH (15 mL) and reflux for 1 h. The formation of a precipitate wasobserved. After the reaction mixture was cooled down to r.t., theprecipitate was collected by filtration, partitioned between NaOH 1N (10mL) and AcOEt (10 mL) and the aqueous phase was extracted twice withAcOEt (10 mL). The organic layers were dried over Na₂SO₄, filtered andevaporated under reduced pressure to provide the desired compound 19(77% yield). 1H NMR (300 MHz, CD₃OD) δ 8.38 (s, 1H), 7.89 (s, 2H), 4.36(s, 2H), 3.41 (t, 2H, J=6.0 Hz), 3.21 (t, 2H, J=6.0 Hz).

tert-butyl8-bromo-10-chloro-3,4-dihydrobenzo[b][1,6]naphthyridine-2(1H)-carboxylate,20

Di-tert-butyl dicarbonate (0.7 mmol) was added to a solution of 19 (0.84mmol) in THF (5 mL) at 0° C. The reaction was stirred at r.t. for 1 h.THF was evaporated off, the residue was partitioned between DCM (25 mL)and NaHCO₃ saturated sol. (25 mL). The aqueous portion was extractedwith DCM (2×25 mL) and the organic layers were dried over Na₂SO₄,filtered and evaporated under reduced pressure to obtain theintermediate 20 (80% yield). MS ESI (m/z) 397 (M+H)⁺. ¹H NMR (300 MHz,CDCl₃) δ 8.34 (d, 1H, J=2.1 Hz), 7.86 (d, 1H, J=9.0 Hz), 7.77 (dd, 1H,J₁=2.1, J₂=8.7 Hz), 4.82 (s, 2H), 3.83 (t, 2H, J=6.0 Hz), 3.16 (t, 2H,J=6.0 Hz), 1.52 (s, 9H).

tert-butyl8-bromo-10-((3-chloro-4-methoxybenzyl)amino)-3,4-dihydrobenzo[b][1,6]naphthyridine-2(1H)-carboxylate,21

A mixture of 20 (0.075 mmol), 3-chloro-4-methoxybenzyl aminehydrochloride (0.38 mmol), TEA (0.38 mmol) and NaI (0.0037 mmol) in NMP(1 mL) was heated to 130° C. and stirred overnight. The reaction wasdiluted with Et₂O (10 mL) and washed with H₂O (2×20 mL) and brine (20mL). The organic layers were dried over Na₂SO₄, filtered and evaporatedunder reduced pressure to give the desired intermediate 21 (40% yield).¹H NMR (300 MHz, CDCl₃) δ 8.08 (s, 1H), 7.84 (s, 1H), 7.68 (d, 1H, J=9.0Hz), 7.35 (s, 1H), 7.19 (d, 1H, J=8.1 Hz), 6.93 (d, 1H, J=8.1 Hz), 4.57(s, 4H), 3.91 (s, 3H), 3.76 (t, 2H, J=6.0 Hz), 3.19 (t, 2H, J=5.7 Hz),1.49 (s, 9H).

8-bromo-N-(3-chloro-4-methoxybenzyl)-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridin-10-amine,F

A solution of Et₂O/HCl 2M was added dropwise to a solution of 21 inDCM:Dioxane (1:2) (1.5 mL). The mixture was stirred at r.t. overnight.The reaction mixture was diluted with HCl 1N and the organic layer wasdiscarded. The aqueous layer was then basified by using NaHCO₃ andextracted with DCM (3×10 mL). The organic layers were dried over Na₂SO₄,filtered and evaporated under reduced pressure. Flash chromatography(DCM:MeOH 1:1) gave the desired product (15% yield). MS ESI (m/z) 432(M+H)⁺. ¹H NMR (300 MHz, CDCl₃) δ 8.04 (s, 1H), 7.84-7.73 (m, 2H),7.65-7.62 (m, 2H), 7.33 (s, 1H), 7.14 (d, 1H, J=8.4 Hz), 6.89 (d, 1H,J=8.4 Hz), 4.45 (d, 2H, J=4.5 Hz), 3.96 (s, 2H), 3.90 (s, 3H), 3.22 (t,2H, J=6.0 Hz), 3.04 (t, 2H, J=6.0 Hz).

Example 710-((3-chloro-4-methoxybenzyl)amino)-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

2-benzyl-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,22

A mixture of 3 (12.3 mmol) and 1-benzylpiperidin-4-one (12.3 mmol) inPOCl₃ (50 mL) was heated at 60° C. for 6 h. The excess of POCl₃ wasevaporated off; the residue was treated with iced H₂O and NaOH 10% (50mL) and extracted with CH₂Cl₂ (3×50 mL). The organic layers were driedover Na₂SO₄, filtered and evaporated under reduced pressure. The residuewas purified by flash chromatography (Hex: AcOEt, 1:2) to give thedesired product (54% yield). MS ESI (m/z) 334 (M+H)⁺; ¹H NMR (300 MHz,CDCl₃) δ 8.56 (d, 1H, J=1.8 Hz), 8.04 (d, 1H, J=8.7 Hz), 7.82 (dd, 1H,J₁=1.8, J₂=8.7 Hz), 7.43-7.31 (m, 5H), 3.93 (s, 2H), 3.83 (s, 2H), 3.26(t, 2H, J=6.0 Hz), 2.92 (t, 2H, J=6.0 Hz).

10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,23

1-chloroethyl chloroformate (9.0 mmol) was added dropwise to a solutionof 22 (6.0 mmol) in DCE (15 mL) at 0° C. The mixture was heated toreflux for 2 h. DCE was evaporated off and the residue was dissolved inMeOH (15 mL) and reflux for 1 h. The formation of a precipitate wasobserved. After the reaction mixture was cooled down to r.t., theprecipitate was collected by filtration and partitioned between NaOH 1N(50 mL) and AcOEt (50 mL) and the aqueous layer was extracted twice withAcOEt (50 mL). The organic layers were dried over Na₂SO₄, filtered andevaporated under reduced pressure to yield the intermediate 23 (62%yield). MS ESI (m/z) 244 (M+H)⁺; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, 1H,J=1.2 Hz), 8.04 (d, 1H, J=8.7 Hz), 7.82 (dd, 1H, J₁=1.8, J₂=8.4 Hz),4.28 (s, 2H), 3.30 (t, 2H, J=6.0 Hz), 3.17 (t, 2H, J=6.0 Hz), 1.73 (s,1H).

tert-butyl10-chloro-8-cyano-3,4-dihydrobenzo[b][1,6]naphthyridine-2(1H)-carboxylate,24

Di-tert-butyl dicarbonate (3.5 mmol) was added to a solution of 23 (3.5mmol) in DCM (10 mL) at 0° C. The reaction was stirred at r.t. for 1 h.The reaction was washed with NaHCO₃ saturated solution (2×30 mL) and theorganic layer was dried over Na₂SO₄, filtered and evaporated underreduced pressure to obtain the intermediate 24 (94% yield). MS ESI (m/z)344 (M+H)⁺.

tert-butyl10-((3-chloro-4-methoxybenzyl)amino)-8-cyano-3,4-dihydrobenzo[b][1,6]naphthyridine-2(1H)-carboxylate,25

A mixture of 24 (0.58 mmol), 3-chloro-4-methoxybenzyl aminehydrochloride (2.90 mmol), TEA (2.90 mmol) and NaI (0.03 mmol) in NMP (3mL) was heated to 130° C. and stirred overnight. The reaction wasdiluted with Et₂O (20 mL) and washed with H₂O (2×20 mL) and brine (20mL). The organic layers were dried over Na₂SO₄, filtered and evaporatedunder reduced pressure. Flash chromatography (Hex:AcOEt 1:2) gave thedesired intermediate 25 (49% yield). MS ESI (m/z) 479 (M+H); ¹H NMR (300MHz, CDCl₃) δ 8.33 (d, 1H, J=1.5 Hz), 7.94 (d, 1H, J=8.7 Hz), 7.70 (dd,1H, J₁=1.8, J₂=8.7 Hz), 7.31 (s, 1H), 7.17 (dd, 1H, J₁=2.1, J₂=8.4 Hz),6.90 (d, 1H, J=8.1 Hz), 4.60-4.55 (m, 4H), 4.36 (s, 1H), 3.89 (2, 3H),3.75 (t, 2H, J=6.0 Hz), 3.12 (t, 2H, J=6.0 Hz), 1.47 (s, 9H).

10-((3-chloro-4-methoxybenzyl)amino)-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,G

A solution of Et₂O/HCl 2M was added dropwise to a solution of 25 inDCM:Dioxane (1:2) (1.0 mL). The mixture was stirred at r.t. overnight.The reaction mixture was diluted with HCl 1N and the organic layer wasdiscarded. The aqueous layer was then basified by using NaHCO₃ andextracted with DCM (3×10 mL). The organic layers were dried over Na₂SO₄,filtered and evaporated under reduced pressure. Flash chromatography(DCM:MeOH 1:1) gave the desired product (20% yield). MS ESI (m/z) 379(M+H)⁺; ¹H NMR (300 MHz, CDCl₃) δ 8.32 (s, 1H), 7.97 (d, 1H, J=9.7 Hz),7.71 (d, 1H, J=9.0 Hz), 7.33 (d, 1H, J=1.8 Hz), 7.17 (d, 1H, J=8.4 Hz),6.93 (d, 1H, J=8.7 Hz), 4.56 (d, 2H, J=5.4 Hz), 4.09 (s, 1H), 3.97 (s,2H), 3.92 (s, 3H), 3.25 (t, 2H, J=6.0 Hz), 3.11 (t, 2H, J=6.0 Hz).

Example 82-acetyl-10-[(3-chloro-4-methoxybenzyl)amino]-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile

2-acetyl-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,26

Acetic anhydride (0.41 mmol) was slowly added to a stirring solution of23 (0.205 mmol; prepared as above) in DCM (1 mL) at 0° C. The reactionwas stirred at r.t. for 1 h. Then iced-water was added to the reactionand the organic layer was washed with H₂O (2×5 mL) and NaHCO₃ (5 mL),dried over Na₂SO₄, filtered and evaporated under reduced pressure togive the compound 26 (77% yield). MS ESI (m/z) 286 (M+H)⁺; ¹H NMR (300MHz, CDCl₃) δ 8.59 (s, 2H), 8.12-8.06 (m, 2H), 7.88-7.84 (m, 2H), 5.01(s, 2H, 62%), 4.87 (s, 2H, 38%), 4.01 (t, 2H, J=6.0 Hz, 32%), 3.88 (t,2H, J=6.0 Hz, 68%), 3.30-3.21 (m, 4H), 2.27-2.25 (m, 6H).

2-acetyl-10-[(3-chloro-4-methoxybenzyl)amino]-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine-8-carbonitrile,H

A mixture of 26 (0.14 mmol), 3-chloro-4-methoxybenzyl aminehydrochloride (0.70 mmol), TEA (0.70 mmol) and NaI (0.007 mmol) in NMP(2 mL) was heated to 130° C. and stirred overnight. The mixture wasdiluted with CH₂Cl₂ (10 mL) and washed with H₂O (2×10 mL) and brine (10mL). The organic layers were dried over Na₂SO₄, filtered and evaporatedunder reduced pressure. Flash Chromatography (AcOEt: MeOH 9:1) gave thedesired product (20% yield). MS ESI (m/z) 421 (M+H)⁺; ¹H NMR (300 MHz,CDCl₃) δ 8.34 (s, 1H), 7.96 (d, 1H, J=8.7 Hz), 7.73 (dd, 1H, J₁=1.5,J₂=9.0 Hz), 7.31 (d, 1H, J=2.1 Hz), 7.20 (dd, 1H, J₁=2.1, J₂=8.4 Hz),6.93 (d, 1H, J=8.1 Hz), 4.70 (s, 2H), 4.64 (d, 2H, J=6.0 Hz), 4.42 (s,1H), 3.91 (s, 3H), 3.82 (t, 2H, J=6.0 Hz), 3.20 (t, 2H, J=6.0 Hz), 2.21(s, 3H).

Example 9 PDE5 Inhibition Assay

Materials and Methods: PDE5 inhibition was assayed at BPS Bioscience(San Diego, Calif.) using BPS PDE assay kits (BPS Catalog number 60300,enzyme lot 090810). Test compounds were supplied as liquid solutions of10 mM concentration in DMSO. Sildenafil, used as a standard wasdissolved in DMSO. Intermediate dilutions were 10% DMSO in PDE assaybuffer and tests at ranges from 0.1 to 100 nM. 0.1 ng of enzyme was usedper reaction, and the substrate was 100 nM FAM-cGMP.

Assay conditions: A series of dilutions of the test compounds wereprepared with 10% DMSO in assay buffer and 5 μl of the dilution wasadded to a 50 μl reaction so that the final concentration of DMSO is 1%in all of reactions. The enzymatic reactions were conducted at roomtemperature for 60 minutes in a 50 μl mixture containing PDE assaybuffer, 100 nM FAM-cAMP or 100 nM FAM-cGMP, a PDE enzyme, and the testcompound. After the enzymatic reaction, 100 μl of a binding solution(1:100 dilution of the binding agent with the binding agent diluent) wasadded to each reaction and the reaction was performed at roomtemperature for 60 minutes. Fluorescence intensity was measured at anexcitation of 485 nm and an emission of 528 nm using a Tecan Infinite M1000 microplate reader.

Data analysis: PDE activity assays were performed in duplicate at eachconcentration.

Fluorescence intensity is converted to fluorescence polarization usingthe Tecan Magellan6 software. The fluorescence polarization data wereanalyzed using the computer software, Graphpad Prism. The fluorescencepolarization (FP_(t)) in absence of the compound in each data set wasdefined as 100% activity. In the absence of PDE and the compound, thevalue of fluorescent polarization (FP_(b)) in each data set was definedas 0% activity. The percent activity in the presence of the compound wascalculated according to the following equation: %activity=(FP−FP_(b))/(FP_(t)−FP_(b))×100%, where FP=the fluorescencepolarization in the presence of the compound.

The values of % activity versus a series of compound concentrations werethen plotted using non-linear regression analysis of Sigmoidaldose-response curve generated with the equationY=B+(T−B)/1+10^(((LogEC50−X)×Hill Slope)), where Y=percent activity,B=minimum percent activity, T=maximum percent activity, X=logarithm ofcompound and Hill Slope=slope factor or Hill coefficient. The IC50 valuewas determined by the concentration causing a half-maximal percentactivity.

Compounds exhibited PDE5 inhibition in the nanomolar range or below.Exemplary inhibition of representative compounds is shown in Table 1.

TABLE 1 PDE5 and PDE6 Inhibition of Representative Compounds. CompoundPDE5 IC₅₀ (nM) PDE6 IC₅₀ (nM) A 21.6 B 1.0 C 0.2 D 0.07 30.0 E 43.8 6617F 5.4 G 1.55 >100 H 0.056 30.1

Example 10 Hippocampal cGMP Assay

2-3 month old male and female mice (20-25 g; C57B16 mice) were injectedwith compound H (3 mg/kg and 10 mg/Kg, 2% DMSO & 2% Tween, i.p.) orVehicle (2% DMSO & 2% Tween, i.p.). 30 min after administration ofvehicle or compound H, mice were subjected to foot shock and sacrificed10 sec, 1 min and 3 min aftershock by cervical dislocation anddecapitation. The hippocampal samples were extracted and snap frozen inliquid nitrogen. Levels of cGMP were quantitated by Enzyme Immunoassayprocedure (Cayman Chemical Company, Item no. 581021) following themanufacturer's guidelines in duplicate. cGMP levels were normalized withthe protein concentration calculated using BCA Protein Assay Reagent(Thermo Scientific).

cGMP levels in adult mice after treatment with compound H were measured.In a series of preliminary experiments, foot shock induces an immediateincrease in cGMP levels in the hippocampus (FIG. 1). Concentration ofcGMP was measured by enzyme immunoassay. Basal represents cGMP levelswithout foot shock. Values are the mean of duplicate determinations.Error bars show S.E.M. (n=3 per group); *p<0.01; ^(‡)p=0.033. Compound H(3 mg/kg and 10 mg/Kg, i.p., 30 minutes prior to electric shock) furtherenhanced cGMP levels at 10 sec, 60 sec, and 180 sec (0.48±0.014,0.58±0.033, and 0.58±0.044 pmol/mg, respectively, at the concentrationof 3 mg/kg; 0.56±0.033, 0.70±0.028, and 0.63±0.013 pmol/mg,respectively, at the concentration of 10 mg/Kg) as compared to vehicle(0.32±0.015, 0.41±0.028, and 0.46±0.038 pmol/mg after 10 sec, 60 sec and3 min, respectively).

Example 11 Long-Term Potentiation Electrophysiological StudiesExperimental Section

Transverse hippocampal slices (400 μm) were cut with a tissue chopper(EMS, PA) and maintained in an interface chamber at 29° C. for 90 minprior to recording, as described in Vitolo et al, Proc. Natl. Acad. Sci.USA 2002, 13217-13221, herein incorporated by reference in its entirety.The extracellular bath solution consisted of 124.0 mM NaCl, 4.4 mM KCl,1.0 mM Na₂HPO₄, 25.0 mM NaHCO₃, 2.0 mM CaCl₂, 2.0 mM MgSO₄, and 10.0 mMglucose, continuously aerated with 95% O₂/5% CO₂ to a final pH of 7.4.Field extracellular postsynaptic responses (fEPSPs) were recorded byplacing the stimulating and recording electrodes in CA1 stratumradiatum. A bipolar tungsten electrode (FHC, Bowdoin, Me.) was used as astimulating electrode, and a glass pipette filled with bath solution wasused as a recording electrode. Basal synaptic transmission was firstassessed by plotting the stimulus voltages (V) against slopes of fEPSPto generate input-output relations. A 20-30 min baseline was firstrecorded every minute at an intensity that evoked a response atapproximately 35% of the maximum evoked response. LTP was induced usinga theta-burst stimulation (4 pulses at 100 Hz, with the bursts repeatedat 5 Hz, and each tetanus consisting of 3 ten-burst trains separated by15 sec). Responses were measured as fEPSP slopes expressed as percentageof baseline.

It was determined whether compound H (50 nM, through the bath perfusion,for 5 min prior to the tetanus) rescues the defect in LTP in 3-4 monthold APP/PS1 animals. 201.76%±14.09 potentiation was found in slices fromtransgenic mice treated with compound compared to 137.30%±10.83potentiation in slices from APP/PS1 mice treated with vehicle (FIG. 2).Compound H rescues the defect in LTP of APP/PS1 slices. Residualpotentiation recorded during the last 5 minutes of a 2 hr recordingfollowing tetanic stimulation of the Schaffer collateral fibers at theCA3-CA1 connection. The compound had no effect onto WT slices. P<0.05comparing compound-treated slices vs vehicle-treated slices in APP/PS1mice.

Preliminary studies with sildenafil have demonstrated that PDE5inhibition has prolonged beneficial effects on synaptic and cognitiveabnormalities in APP/PS1 mice that persist beyond the administration ofthe inhibitor. This finding suggests the possibility of using thesedrugs to interfere with the progression of the memory deficits. It wasdecided to investigate whether this important therapeutic possibilityoccurs with compound H. In these experiments, both APP/PS1 and WT miceof 3 months of age were i.p. injected with 3 mg/kg/day with compound Hfor 3 weeks, then the treatment was stopped for 9-12 weeks prior totesting. Slices from transgenic mice treated with the compound had226.23%±6.72 potentiation compared to 164.12%±10.37 in slices fromvehicle-treated transgenic mice (FIG. 3). Residual potentiation recordedduring the last 5 minutes of a 2 hr recording following tetanicstimulation of the Schaffer collateral fibers at the CA3-CA1 connection.Daily treatment with compound H (3 mg/kg, i.p.) for 3 weeks at the ageof 3-4 months re-established normal potentiation when slices wererecorded at 6-7 months of age.

Example 12 Behavior Studies Experimental Section

The radial arm water maze task, a hybrid of the classic Morris WaterMaze and the radial arm land maze, was performed as described in J.Alamed, D. M. Wilcock, D. M. Diamond, M. N. Gordon, D. Morgan, Two-dayradial-arm water maze learning and memory task; robust resolution ofamyloid-related memory deficits in transgenic mice, Nat. Prot. 1 (2006)1671-1679, herein incorporated by reference in its entirety. The mousehad to swim in 6 alleys (arms) radiating from a central area until itfound a hidden (submerged) platform at the end of one of the arms, basedon visual cues placed in the room. The first day of the protocol was atraining day on which mice were trained to identify the platformlocation by alternating between a visible and a hidden platform in agoal arm. The final 3 trials on that day and all 15 trials on day 2 useda hidden escape platform to force mice to use spatial cues to identifythe location of the goal arm. To avoid learning limitations imposed byexhausting practice and to avoid fatigue that may result fromconsecutive trials, spaced practice training was established by runningthe mice in cohorts of 4 and alternating different cohorts through the15 training trials over 3-hour testing periods each day. The number ofincorrect arm entries (entries to arms with no platform) was counted. Ifthe animal entered the incorrect arm it was gently pulled back to thestart arm. Failure to select an arm after 15 sec was counted as an errorand the mouse was returned to the start arm. Each trial lasted up to 1min. After 1 min, if the platform had not been located, the mouse wasguided gently through the water by placing a hand behind it to direct ittowards the platform. The mouse rested on the platform for 15 sec. Thegoal platform location was different for each mouse. On day 2, the sameprocedure was repeated as on day 1 for all 15 trials using only thehidden platform. For data analysis, averages for each mouse werecalculated using blocks of 3 trials.

Fear conditioning was assessed as described in B. Gong, O. V. Vitolo, F.Trinchese, S. Liu, M. Shelanski, O. Arancio, Persistent improvement insynaptic and cognitive functions in an Alzheimer mouse model afterrolipram treatment, J. Clin. Invest. 114 (2004) 1624-1634; F. Trinchese,S. Liu, F. Battaglia, S. Walter, P. M. Mathews, O. Arancio, Progressiveage-related development of Alzheimer-like pathology in APP/PS1 mice,Ann. Neurol. 55 (2004) 801-814; and B. Gong, Z. Cao, P. Zheng, O. V.Vitolo, S. Liu, A. Staniszewski, D. Moolman, H. Zhang, M. Shelanski, O.Arancio, Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induceddecreases in synaptic function and contextual memory, Cell 126 (2006)775-788; each herein incorporated by reference in its entirety. First,sensory perception of electric foot shock was examined in differentgroups of mice through the threshold assessment test. Animals wereplaced in the conditioning chamber and the electric current (0.1 mA for1 sec) was increased at 30 s intervals from 0.1 mA to 0.7 mA. Thresholdto flinching (first visible response to shock), jumping (first extrememotor response), and vocalized response were quantified for each animalby averaging the shock intensity at which each animal showed thebehavioral response to that type of shock. Training of fear conditioningwas performed by placing the mouse in a conditioning chamber for 2 minbefore the onset of a tone (Conditioned Stimulus (CS), 30 sec, 85 dBsound at 2800 Hz). In the last 2 sec of the CS, mice were given a 2 sec,0.7 mA mild foot shock (Unconditioned Stimulus, (US)) through the barsof the floor. After the US, the mice were left in the chamber foranother 30 s. Freezing behavior, defined as the absence of movementsexcept for respiratory excursions, was scored using Freezeview software(Med Associates, St. Albans, Vt.). Contextual fear learning wasevaluated 24 hrs after training by measuring freezing responses for 5min in the same chamber where the mice were trained. Cued fear learningwas evaluated 24 hrs after contextual testing. The mice were placed in anovel context for 2 min (pre-CS test), after which they were given a CSfor 3 min (CS test), and freezing behavior was measured during the first30 sec that mimic the CS-US conditioning and the remaining 2.5 min.

It was determined whether compound H (3 mg/kg, i.p, for 3 weeks) rescuesthe defect in reference memory in 3-4 month old APP/PS1 animals.Transgenic mice treated with the compound made between 1 to 2 errors inthe last trial of the 2-day RAWM test in contrast to transgeniclittermates treated with vehicle that made about 3 errors (FIG. 4).Compound H rescues the defect in reference memory of APP/PS1 mice. Dailytreatment with the compound for 3 weeks at the age of 3-4 months reducedthe number of errors with the 2-day radial arm water maze in APP/PS1mice. The compound had no effect on WT mice. p<0.05 comparingcompound-treated transgenic mice vs vehicle-treated APP/PS1 mice.

It was also determined if compound H (3 mg/kg, i.p, for 3 weeks) rescuesthe defect in fear memory in 3-4 month old APP/PS1 animals. Transgenicmice treated with the compound froze ˜30% of the time when they wereexposed after 24 hrs to the same context in which they had received anelectric shock compared to about 15% for vehicle-treated APP/PS1 mice(FIG. 5). Compound H rescues the defect in fear memory of APP/PS1 mice.Daily treatment with the compound for 3 weeks at the age of 3-4 monthsre-established normal freezing in a test for contextual fear memory inAPP/PS1 mice. The compound had no effect on WT mice. p<0.05 comparingcompound-treated transgenic mice vs vehicle-treated APP/PS1 mice.

The possibility of compound H to interfere with the progression ofmemory deficits was investigated. Both APP/PS1 and WT mice of 3-4 monthsof age were i.p. injected with compound H (3 mg/kg/day) for 3 weeks,then the treatment was stopped for 9-12 weeks prior to testing. Micewere next subjected to 2-day RAWM and fear conditioning. Transgenic micetreated with the compound made ˜1 error in the 2-day RAWM test comparedto ˜2 errors in vehicle-treated transgenic mice (FIG. 6). Dailytreatment with compound H (3 mg/kg, i.p.) for 3 weeks at the age of 3-4months reduced the number of errors with the 2-day radial arm water mazewhen mice were examined at 6-7 months of age. Contextual memoryexperiments revealed ˜40% freezing in transgenic mice treated with thecompound compared to ˜15% in vehicle-treated transgenic mice (FIG. 7).Daily treatment with compound H (3 mg/kg, i.p.) for 3 weeks at the ageof 3-4 months re-established normal freezing in a test for contextualfear memory when mice were examined at 6-7 months of age. Thus, synapticand cognitive improvements persist beyond the administration of theinhibitor.

Example 13 Pharmacokinetic Studies

The PK study of compound H (25 mg/kg, i.p.) was conducted in male C57B16mice. The plasma and brain concentrations of compound H at differenttime points are shown in FIG. 8.

Following i.p. administration of 25 mg/kg to C57B16 mice, compound H wasrapidly absorbed as indicated by peak plasma concentration occurring at0.25 h after dosing (Table 2). The distribution of compound H to brainwas fast with a similar T_(max) value in the brain. The exposure ofcompound H in brain was lower than in plasma with a C_(max) ratio of0.31. The elimination half-lives of compound H in brain and plasma were0.65 and 1.07 h, respectively.

TABLE 2 Pharmacokinetic parameters for Compound H in mice. RatioParameters Brain Plasma (brain/plasma) Tmax (h) 0.25 0.25 — Cmax (ng/mLor ng/g) 492.25 1571.44 0.313 t½ (h) 0.65 1.07 — MRT (h) 0.95 1.56 —C_(max): the maximum observed concentration; T_(max): time correspondingto C_(max); MRT: Mean Residence Time; t_(1/2): the eliminationhalf-life.

Example 14 Acute Toxicity Studies

Mice were given compound H at two different concentrations (120 or 240mg/kg, i.p.) or vehicle, and sacrificed afterwards (compound H at aconcentration of 480 mg/kg, i.p. was fatal, however the efficacious doseof compound H was 3 mg/kg). Brain, muscle, kidney, and liver wereharvested from each mouse, and tissue from each organ was examinedhistologically by a board-certified pathologist for signs of toxicity.Sections of brain did not show any signs of toxicity by H&E, and a GFAPimmunostain showed no signs of gliosis (FIG. 9). Additional stains onsections of brain with LFB-PAS revealed no signs of demyelination, andan immunostain for CD45 showed no signs of inflammation. Sections ofmuscle showed no signs of toxicity by H&E, and a trichrome stain showedno muscle fibrosis. Sections of kidney showed no toxicity by H&Estaining. Additional staining of kidney sections with PAS did not showany abnormalities in the glomerular basement membrane, and trichromestaining showed no fibrosis. Sections of liver did not show any signs oftoxicity by H&E staining, and a trichrome stain showed no fibrosis orcirrhotic liver disease. The compound showed no signs of neurotoxicityin the brain. H&E staining shows no difference between the three groups(FIG. 9A: vehicle; FIG. 9B: 120 mg/kg; FIG. 9C: 240 mg/kg). In addition,a GFAP immunostain shows no signs of gliosis in any of the three groups(FIG. 9D: vehicle; FIG. 9E: 120 mg/kg, FIG. 9F: 240 mg/kg).

Example 151-(10-((3-chloro-4-methoxybenzyl)amino)-3,4-dihydrobenzo[b][1,6]naphthyridin-2(1H)-yl)ethan-1-one

2-benzyl-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine 28

A mixture of 2-aminobenzoic acid 27 (7.3 mmol) and1-methylpiperidin-4-one (7.3 mmol) in POCl3 (5 mL) was heated at 60° C.for 6 h. The excess POCl₃ was evaporated off; the residue was treatedwith iced H₂O and NaHCO₃ and extracted with CH₂Cl₂ (3×25 mL). Theorganic layer was dried over Na₂SO₄, filtered and evaporated underreduced pressure. Purification by Flash Chromatography (5% MeOH inAcOEt) gave the desired compound 28. MS ESI (m/z) 309 (M+H)⁺; ¹H NMR(400 MHz, CDCl₃) δ 8.16 (dd, 1H, J₁=1.2, J₂=8.4 Hz), 8.0 (d, 1H, J=8.4Hz), 7.71-7.67 (m, 1H), 7.58-7.54 (m, 1H), 7.44-7.42 (m, 2H), 7.39-7.35(m, 2H), 7.33-7.30 (m, 1H), 3.95 (s, 2H), 3.84 (s, 2H), 3.25 (t, 2H,J=5.6 Hz), 2.93 (t, 2H, J=5.6 Hz).

10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine 29

1-chloroethyl chloroformate (6.3 mmol) was added dropwise to a solutionof 28 (4.2 mmol) in DCE (5 mL) at 0° C. The mixture was heated to refluxfor 2 h. DCE was evaporated off and the residue was dissolved in MeOH (5mL) and reflux for 1 h. The formation of a precipitate was observed.After the reaction mixture was cooled down to r.t., the precipitate wascollected by filtration and partitioned between NaOH 1N (50 mL) andAcOEt (50 mL) and the aqueous layer was extracted twice with AcOEt (50mL). The organic layers were dried over Na₂SO₄, filtered and evaporatedunder reduced pressure to yield 29. MS ESI (m/z) 219 (M+H)⁺.

1-(10-chloro-3,4-dihydrobenzo[b][1,6]naphthyridin-2(1H)-yl)ethan-1-one30

Acetic anhydride (3.65 mmol) was slowly added to a stirring solution of29 (1.83 mmol) in DCM (4 mL) at 0° C. The reaction was stirred at r.t.for 1 h. Then iced-water was added to the reaction and the organic layerwas washed with H₂O (2×10 mL) and NaHCO₃ (10 mL), dried over Na₂SO₄,filtered and evaporated under reduced pressure to give compound 30.

1-(10-((3-chloro-4-methoxybenzyl)amino)-3,4-dihydrobenzo[b][1,6]naphthyridin-2(1H)-yl)ethan-1-one,I

A solution of 30 (1.15 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (3.45 mmol), NaI (0.06 mmol), and triethylamine (3.45mmol) in NMP (5.0 mL) was heated at 130° C. for 6 h. The solvent wasevaporated off and the residue was purified by Flash Chromatography(AcOEt: MeOH 8:2) to give I. MS ESI (m/z) 396 (M+H); ¹H NMR (400 MHz,CDCl₃) δ 8.05-8.02 (m, 1H), 7.92 (d, 1H, J=9.2 Hz), 7.67-7.63 (m, 1H),7.45-7.40 (m, 1H), 7.35 (d, 1H, J=2.4 Hz), 7.22 (dd, 1H, J₁=2.0, J₂=8.4Hz), 6.92 (d, 1H, J=8.4 Hz), 4.75 (s, 2H), 4.65 (d, 2H, J=5.2 Hz), 3.90(s, 4H), 3.81 (t, 2H, J=6.0 Hz), 3.27 (t, 2H, J=6.4 Hz), 2.20 (s, 3H).

Example 161-(10-((3-chloro-4-methoxybenzyl)amino)-8-methoxy-3,4-dihydrobenzo[b][1,6]naphthyridin-2(1H)-yl)ethan-1-one

2-benzyl-10-chloro-8-methoxy-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine62

A mixture of 2-amino-5-methoxybenzoic acid 61 (1.2 mmol) and1-methylpiperidin-4-one (1.2 mmol) in POCl₃ (2.0 mL) was heated at 60°C. for 3 h. The excess POCl₃ was evaporated off; the residue was treatedwith iced NH₃ aq. and extracted with AcOEt (3×20 mL). The organic layerwas dried over Na₂SO₄, filtered and evaporated under reduced pressure.Purification by Flash Chromatography (AcOEt) gave the desired compound62. MS ESI (m/z) 339 (M+H)⁺; H NMR (400 MHz, CDCl₃) δ 7.86 (d, 1H, J=9.2Hz), 7.41-7.23 (m, 7H), 3.93 (s, 3H), 3.90 (s, 2H), 3.80 (s, 2H), 3.18(t, 2H, J=6.0 Hz), 2.87 (t, 2H, J=5.6 Hz).

10-chloro-8-methoxy-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridine 63

1-chloroethyl chloroformate (0.62 mmol) was added dropwise to a solutionof 62 (0.41 mmol) in DCE (3.0 mL) at 0° C. The mixture was heated toreflux for 2 h. DCE was evaporated off and the residue was dissolved inMeOH (5.0 mL) and reflux for 1 h. The formation of a precipitate wasobserved. After the reaction mixture was cooled down to r.t., theprecipitate was collected by filtration and partitioned between NaOH 1N(20 mL) and AcOEt (20 mL) and the aqueous layer was extracted twice withAcOEt (20 mL). The organic layers were dried over Na₂SO₄, filtered andevaporated under reduced pressure to yield 63. MS ESI (m/z) 249 (M+H)⁺;¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, 1H, J=8.8 Hz), 7.39 (d, 1H, J=2.8Hz), 7.34 (dd, 1H, J₁=2.4, J₂=8.8 Hz), 4.25 (s, 2H), 3.96 (s, 3H), 3.28(t, 2H, J=6.0 Hz), 3.11 (t, 2H, J=6.4 Hz).

1-(10-chloro-8-methoxy-3,4-dihydrobenzo[b][1,6]naphthyridin-2(1H)-yl)ethan-1-one64

Acetic anhydride (0.56 mmol) was slowly added to a stirring solution of63 (0.28 mmol) in DCM (1.0 mL) at 0° C. The reaction was stirred at r.t.for 1 h. Then iced-water was added to the reaction and the organic layerwas washed with H₂O (2×10 mL) and NaHCO₃ (10 mL), dried over Na₂SO₄,filtered and evaporated under reduced pressure to give compound 64. MSESI (m/z) 291 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, 1H, J=9.2 Hz),7.42-7.40 (m, 1H), 7.39-7.36 (m, 1H), 4.98 (s, 2H), 3.97 (s, 3H), 3.86(t, 2H, J=6.0 Hz), 3.22 (t, 2H, J=6.0 Hz), 2.25 (s, 3H).

1-(10-((3-chloro-4-methoxybenzyl)amino)-8-methoxy-3,4-dihydrobenzo[b][1,6]naphthyridin-2(1H)-yl)ethan-1-oneJ

A solution of 64 (0.15 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.69 mmol), NaI (0.007 mmol), and triethylamine (0.69mmol) in NMP (3.0 mL) was heated at 130° C. for 6 h. The solvent wasevaporated off and the residue was purified by Flash Chromatography(AcOEt: MeOH 8:2) to give product J. MS ESI (m/z) 426 (M+H)⁺. ¹H NMR(400 MHz, CDCl₃) δ 7.86 (d, 1H, J=8.8 Hz), 7.37 (d, 1H, J=2.0 Hz), 7.29(dd, 1H, J₁=2.8, J₂=9.6 Hz), 7.23 (dd, 1H, J₁=2.4, J₂=8.0 Hz), 7.15 (d,1H, J=2.8 Hz), 6.91 (d, 1H, J=8.4 Hz), 4.73 (s, 2H), 4.53 (d, 2H, J=5.6Hz), 3.90 (s, 4H), 3.80 (t, 2H, J=6.4 Hz), 3.75 (s, 3H), 3.18 (t, 2H,J=6.0 Hz), 2.21 (s, 3H).

The following general scheme can be used to prepare additional compoundsin accordance with certain aspects of the invention:

Example 179-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-b]quinoline-7-carbonitrile

2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-6-carbonitrile 33

(Cl₃CO)₂CO (3.08 mmol) was added to a suspension of2-amino-5-cyanobenzoic acid 3 (9.25 mmol) in 1,4-dioxane at 0° C. Thehomogeneous reaction mixture was heated to 90° C. for 5 h and thencooled down. The resulting precipitate 33 was isolated by filtration. 1HNMR (400 MHz, DMSO-d₆) δ 12.15 (s, 1H), 8.38 (d, 1H, J=1.6 Hz), 8.11(dd, 1H, J₁=1.6, J₂=8.8 Hz), 7.25 (d, 1H, J=8.4 Hz)

methyl 6-cyano-4-hydroxy-2-methyl-1,4-dihydroquinoline-3-carboxylate 34

NaH (6.38 mmol) was added portionwise to a solution of methylacetoacetate (6.38 mmol) in DMA (2 mL). Compound 33 (5.32 mmol) wasadded and the reaction mixture was stirred to 120° C. for 30 min. Thesolvent was reduced and water was added. The resulting precipitate 34was collected by filtration. 1H NMR (400 MHz, DMSO-d₆) δ 12.25 (s, 1H),8.39 (d, 1H, J=2.0 Hz), 8.01 (dd, 1H, J₁=2.0, J₂=8.8 Hz), 7.66 (d, 1H,J=8.8 Hz), 3.70 (s, 3H), 2.41 (s, 3H)

methyl 4-chloro-6-cyano-2-methylquinoline-3-carboxylate 35

Compound 34 (2.0 mmol) was suspended in POCl₃ (4 mL) and heated to 110°C. for 20 min. the homogeneous reaction mixture was slowly poured intoiced NH₃ aq. The aqueous phase was extracted with AcOEt (3×50 mL). Theorganic layers were dried over Na₂SO₄, filtered and evaporated underreduce pressure to give the desire compound 35. MS ESI (m/z) 261 (M+H)⁺;¹H NMR (400 MHz, CDCl₃) δ 8.61 (d, 1H, J=2.0 Hz), 8.12 (d, 1H, J=8.8Hz), 7.92 (dd, 1H, J₁=1.6, J₂=8.4 Hz), 4.05 (s, 3H), 2.75 (s, 3H).

methyl 2-(bromomethyl)-4-chloro-6-cyanoquinoline-3-carboxylate 36

A solution of 35 (0.14 mmol), N-bromosuccinimide (0.22 mmol) and2,2′-Azobis(2-methylpropionitrile) (0.03 mmol) in CCl₄ (1 mL) was heatedto reflux for 4 h. The reaction mixture was cooled down and the solidwas filtered off. The filtrate was concentrated and the residue waspurified by flash chromatography (hexanes: AcOEt 9:1) to give thedesired product 36.

9-chloro-2-ethyl-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-b]quinoline-7-carbonitrile37

To a solution of 36 (0.15 mmol) in ethanol was added EtNH₂ (2M in THF,0.46 mmol). The reaction mixture was heated to reflux for 2 h. Thesolvent was evaporated off and the residue was purified by flashchromatography (Hexanes:AcOEt 3:7) to give the desired product 37. MSESI (m/z) 272 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.84 (d, 1H, J=2.0 Hz),8.23 (d, 1H, J=1.4 Hz), 7.99 (dd, 1H, J₁=1.6, J₂=8.8 Hz), 4.57 (s, 2H),3.78 (q, 2H, J=7.2 Hz), 1.35 (t, 3H, J=6.8 Hz)

9-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-1-oxo-2,3-dihydro-1H-pyrrolo[3,4-b]quinoline-7-carbonitrile,K

A solution of 37 (0.037 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.055 mmol) and (^(i)pr)₂NEt (0.22 mmol) in n-propanol (1mL) was heated to 90° C. for 1 h. After cooling down, the resultingprecipitate K was collected by filtration. MS ESI (m/z) 407 (M+H)⁺; ¹HNMR (400 MHz, CDCl₃) δ 8.58 (t, 1H, J=6.0 Hz), 8.53 (d, 1H, J=1.6 Hz),7.94 (d, 1H, J=9.2 Hz), 7.75 (dd, 1H, J₁=1.6, J₂=8.4 Hz), 7.42 (d, 1H,J=1.6 Hz), 7.30 (dd, 1H, J₁=2.4, J₂=8.4 Hz), 6.96 (d, 1H, J=8.4 Hz),4.93 (d, 2H, J=5.6 Hz), 4.38 (s, 2H), 3.89 (s, 3H), 3.63 (q, 2H, J=7.2Hz), 1.27 (t, 3H, J=7.2 Hz)

Example 189-((3-chloro-4-methoxybenzyl)amino)-2-ethyl-7-methoxy-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one

6-methoxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione 39

(Cl₃CO)₂CO (1.0 mmol) was added to a suspension of2-amino-5-methoxybenzoic acid 38 (3.0 mmol) in 1,4-dioxane (3 mL) at 0°C. The homogeneous reaction mixture was heated to 90° C. for 2 h andthen cooled down. The resulting precipitate 39 was isolated byfiltration. MS ESI (m/z) 192 (M−1); ¹H NMR (400 MHz, DMSO-d₆) δ 11.61(s, 1H), 7.38 (dd, 1H, J₁=2.8, J₂=8.4 Hz), 7.34 (d, 1H, J=2.4 Hz), 7.11(d, 1H, J=8.4 Hz), 3.80 (s, 3H)

methyl 6-methoxy-2-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate 40

NaH (3.1 mmol) was added portionwise to a solution of methylacetoacetate (3.1 mmol) in DMA (3 mL). Compound 39 (2.6 mmol) was addedand the reaction mixture was stirred to 120° C. for 30 min. The solventwas reduced and water was added. The resulting precipitate 40 wascollected by filtration. 1H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 7.49(d, 1H, J=8.8 Hz), 7.45 (d, 1H, J=2.8 Hz), 7.31 (dd, 1H, J₁=3.2, J₂=8.8Hz), 3.82 (s, 3H), 3.75 (s, 3H), 2.37 (s, 3H)

methyl 4-chloro-6-methoxy-2-methylquinoline-3-carboxylate 41

Compound 40 (1.62 mmol) was suspended in POCl₃ (3 mL) and heated to 110°C. for 30 min. the homogeneous reaction mixture was slowly poured intoiced NH₃ aq. The aqueous phase was extracted with AcOEt (3×50 mL). Theorganic layers were dried over Na₂SO₄, filtered and evaporated underreduce pressure to give the desire compound 41. ¹H NMR (400 MHz, CDCl₃)δ 7.93 (d, 1H, J=9.6 Hz), 7.43-7.41 (m, 2H), 4.03 (s, 3H), 3.97 (s, 3H),2.67 (s, 3H).

methyl 2-(bromomethyl)-4-chloro-6-methoxyquinoline-3-carboxylate 42

A solution of 41 (1.47 mmol), N-bromosuccinimide (2.20 mmol) and2,2′-Azobis(2-methylpropionitrile) (0.3 mmol) in CCl₄ (3 mL) was heatedto reflux for 5 h. The reaction mixture was cooled down and the solidwas filtered off. The filtrate was concentrated and the residue waspurified by flash chromatography (hexanes: AcOEt 9:1) to give thedesired product 42. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, 1H, J=10.4 Hz),7.47-7.44 (m, 2H), 4.77 (s, 2H), 4.07 (s, 3H), 3.99 (s, 3H).

9-chloro-2-ethyl-7-methoxy-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one43

To a solution of 42 (0.17 mmol) in ethanol (1 mL) was added EtNH₂ (2M inTHF, 0.52 mmol). The reaction mixture was heated to reflux for 3 h. Thesolvent was evaporated off and the residue was purified by flashchromatography (Hexanes: AcOEt 3:7) to give the desired product 43. ¹HNMR (400 MHz, CDCl₃) δ 8.01 (d, 1H, J=8.8 Hz), 7.64 (d, 1H, J=2.8 Hz),7.50 (dd, 1H, J₁=2.8, J₂=9.6 Hz), 4.49 (s, 2H), 4.01 (s, 3H), 3.76 (q,2H, J=7.2 Hz), 1.33 (t, 3H, J=7.2 Hz)

9-((3-chloro-4-methoxybenzyl)amino)-2-ethyl-7-methoxy-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-oneL

A solution of 43 (0.11 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.16 mmol) and (^(i)pr)₂NEt (0.66 mmol) in n-propanol(1.5 mL) was heated to 90° C. for 1 h. The solvent was evaporated offand the residue was purified by flash chromatography (AcOEt: MeOH 19:1)to give the final product L. MS ESI (m/z) 412 (M+H)⁺; ¹H NMR (400 MHz,CDCl₃) δ 8.14 (t, 1H, J=6.4 Hz), 7.85 (d, 1H, J=9.6 Hz), 7.48 (d, 1H,J=2.4 Hz), 7.37-7.29 (m, 3H), 6.95 (d, 1H, J=8.8 Hz), 4.95 (d, 2H, J=6.8Hz), 4.38 (s, 2H), 3.90 (s, 3H), 3.66 (q, 2H, J=7.2 Hz), 3.59 (s, 3H),1.28 (t, 3H, J=7.2 Hz)

Example 199-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-7-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one

6-(trifluoromethyl)-2H-benzo[d][1,3]oxazine-2,4(1H)-dione 45

(Cl₃CO)₂CO (1.3 mmol) was added to a suspension of2-amino-5-(trifluoromethyl)benzoic acid 44 (3.9 mmol) in 1,4-dioxane (3mL) at 0° C. The homogeneous reaction mixture was heated to 90° C. for 2h and then cooled down. The resulting precipitate 45 was isolated byfiltration. ¹H NMR (400 MHz, DMSO-d₆) δ 12.09 (s, 1H), 8.14 (d, 1H,J=1.2 Hz), 8.07 (dd, 1H, J₁=1.6, J₂=8.8 Hz), 7.32 (d, 1H, J=8.8 Hz)

methyl2-methyl-4-oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate 46

NaH (3.1 mmol) was added portionwise to a solution of methylacetoacetate (3.1 mmol) in DMA (2 mL). Compound 45 (2.6 mmol) was addedand the reaction mixture was stirred to 120° C. for 30 min. The solventwas reduced and water was added. The resulting precipitate 46 wascollected by filtration. MS ESI (m/z) 286 (M+H)⁺.

methyl 4-chloro-2-methyl-6-(trifluoromethyl)quinoline-3-carboxylate 47

Compound 46 (1.6 mmol) was suspended in POCl₃ (1 mL) and heated to 110°C. for 20 min. the homogeneous reaction mixture was slowly poured intoiced NH₃ aq. The aqueous phase was extracted with AcOEt (3×50 mL). Theorganic layers were dried over Na₂SO₄, filtered and evaporated underreduce pressure to give the desire compound 47. MS ESI (m/z) 304 (M+H)⁺.

methyl2-(bromomethyl)-4-chloro-6-(trifluoromethyl)quinoline-3-carboxylate 48

A solution of 47 (0.43 mmol), N-bromosuccinimide (0.64 mmol) and2,2′-Azobis(2-methylpropionitrile) (0.03 mmol) in CCl₄ (2.0 mL) washeated to reflux for 4 h. The reaction mixture was cooled down and thesolid was filtered off. The filtrate was concentrated and the residuewas purified by flash chromatography (hexanes: AcOEt 9:1) to give thedesired product 48.

MS ESI (m/z) 384 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.58 (d, 1H, J=1.2Hz), 8.22 (d, 1H, J=8.8 Hz), 8.01 (dd, 1H, J₁=2.0, J₂=8.8 Hz), 4.77 (s,2H), 4.06 (s, 3H).

9-chloro-2-ethyl-7-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one49

To a solution of 48 (0.21 mmol) in ethanol was added EtNH₂ (2M in THF,0.63 mmol). The reaction mixture was heated to reflux for 2.5 h. Thesolvent was evaporated off and the residue was purified by flashchromatography (Hexanes:AcOEt 1:1) to give the desired product 49.

MS ESI (m/z) 315 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.76 (s, 1H), 8.26(d, 1H, J=8.8 Hz), 8.03 (dd, 1H, J₁=2.0, J₂=8.8 Hz), 4.57 (s, 2H), 3.79(q, 2H, J=6.8 Hz), 1.35 (t, 3H, J=6.8 Hz)

9-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-7-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-oneM

A solution of 49 (0.095 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.14 mmol) and (^(i)pr)₂NEt (0.57 mmol) in n-propanol(1.0 mL) was heated to 90° C. for 1 h. The solvent was evaporated offand the residue was purified by flash chromatography (AcOEt) to give thefinal product M. MS ESI (m/z) 450 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.58(t, 1H, J=6.0 Hz), 8.46 (s, 1H), 8.0 (d, 1H, J=8.8 Hz), 7.80 (dd, 1H,J₁=2.0, J₂=9.2 Hz), 7.45 (d, 1H, J=2.0 Hz), 7.32 (dd, 1H, J₁=2.4, J₂=8.4Hz), 6.95 (d, 1H, J=8.0 Hz), 4.95 (d, 2H, J=6.0 Hz), 4.41 (s, 2H), 3.90(s, 3H), 3.67 (q, 2H, J=7.2 Hz), 1.3 (t, 3H, J=7.2 Hz)

Example 207-chloro-9-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one

methyl 6-chloro-2-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate 51

NaH (9.1 mmol) was added portionwise to a solution of methylacetoacetate (9.1 mmol) in DMA (2 mL). Compound 50 (7.6 mmol) was addedand the reaction mixture was stirred to 120° C. for 30 min. The solventwas reduced and water was added. The resulting precipitate 51 wascollected by filtration. 1H NMR (400 MHz, DMSO-d₆) δ 12.07 (s, 1H), 7.98(d, 1H, J=2.4 Hz), 7.71 (dd, 1H, J₁=2.4, J₂=8.4 Hz), 7.57 (d, 1H, J=8.8Hz), 3.76 (s, 3H), 2.39 (s, 3H).

methyl 4,6-dichloro-2-methylquinoline-3-carboxylate 52

Compound 51 (3.0 mmol) was suspended in POCl₃ (4 mL) and heated to 110°C. for 20 min. the homogeneous reaction mixture was slowly poured intoiced NH₃ aq. The aqueous phase was extracted with AcOEt (3×50 mL). Theorganic layers were dried over Na₂SO₄, filtered and evaporated underreduce pressure to give the desire compound 52. ¹H NMR (400 MHz,DMSO-d₆) δ 8.21 (d, 1H, J=2.4 Hz), 8.08 (d, 1H, J=9.2 Hz), 7.95 (dd, 1H,J₁=2.4, J₂=8.8 Hz), 4.01 (s, 3H), 2.63 (s, 3H).

methyl 2-(bromomethyl)-4,6-dichloroquinoline-3-carboxylate 53

A solution of 52 (1.5 mmol), N-bromosuccinimide (3.0 mmol) and2,2′-Azobis(2-methylpropionitrile) (0.3 mmol) in CCl₄ (1 mL) was heatedto reflux for 4 h. The reaction mixture was cooled down and the solidwas filtered off. The filtrate was concentrated and the residue waspurified by flash chromatography (hexanes: AcOEt 2:1) to give thedesired product 53. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (d, 1H, J=2.0 Hz),8.04 (d, 1H, J=8.4 Hz), 7.78 (dd, 1H, J₁=2.4, J₂=8.8 Hz), 4.78 (s, 2H),4.08 (s, 3H).

7,9-dichloro-2-ethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one 54

To a solution of 53 (0.41 mmol) in ethanol was added EtNH₂ (2M in THF,1.24 mmol). The reaction mixture was heated to reflux for 2 h. Thesolvent was evaporated off and the residue was purified by flashchromatography (Hexanes: AcOEt 3:7) to give the desired product 54.

MS ESI (m/z) 281 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.43 (d, 1H, J=2.0Hz), 8.07 (d, 1H, J=8.8 Hz), 7.79 (dd, 1H, J₁=2.4, J₂=8.8 Hz), 4.52 (s,2H), 3.78 (q, 2H, J=7.6 Hz), 1.33 (t, 3H, J=7.6 Hz).

7-chloro-9-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-oneN

A solution of 54 (0.18 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.27 mmol) and (^(i)pr)₂NEt (1.08 mmol) in n-propanol (1mL) was heated to 90° C. for 2 h. After cooling down, the resultingprecipitate N was collected by filtration. MS ESI (m/z) 416 (M+H)⁺; ¹HNMR (400 MHz, CDCl₃) δ 8.29 (t, 1H, J=5.6 Hz), 8.14 (d, 1H, J=2.0 Hz),7.86 (d, 1H, J=9.2 Hz), 7.58 (dd, 1H, J₁=2.0, J₂=9.2 Hz), 7.45 (d, 1H,J=1.2 Hz), 7.32 (d, 1H, J=8.8 Hz), 6.95 (d, 1H, J=8.4 Hz), 4.93 (d, 2H,J=6.0 Hz), 4.37 (s, 2H), 3.91 (s, 3H), 3.64 (q, 2H, J=7.6 Hz), 1.28 (t,3H, J=7.2 Hz).

Example 219-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one

methyl 2-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate 56

NaH (7.35 mmol) was added portionwise to a solution of methylacetoacetate (7.35 mmol) in DMA (2.0 mL). N-methylisatoic anhydride 55(6.13 mmol) was added and the reaction mixture was stirred to 120° C.for 30 min. The solvent was reduced and water was added. The resultingprecipitate 56 was collected by filtration. MS ESI (m/z) 218 (M+H)⁺; ¹HNMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 8.05 (d, 1H, J=8.0 Hz),7.69-7.65 (m, 1H), 7.53 (d, 1H, J=8.4 Hz), 7.36 (m, 1H), 3.76 (s, 3H),2.39 (s, 3H).

methyl 4-chloro-2-methylquinoline-3-carboxylate 57

Compound 56 (3.9 mmol) was suspended in POCl₃ (3.0 mL) and heated to110° C. for 30 min. the homogeneous reaction mixture was slowly pouredinto iced NH₃ aq. The aqueous phase was extracted with AcOEt (3×50 mL).The organic layers were dried over Na₂SO₄, filtered and evaporated underreduce pressure to give the desire compound 57. MS ESI (m/z) 236 (M+H)⁺;¹H NMR (400 MHz, CDCl₃) δ 8.4 (d, 1H, J=8.4 Hz), 8.03 (d, 1H, J=8.4 Hz),7.79 (td, 1H, J₁=1.2, J₂=7.2), 7.63 (t, 1H, J=8.4), 4.04 (s, 3H), 2.72(s, 3H).

methyl 2-(bromomethyl)-4-chloroquinoline-3-carboxylate 58

A solution of 57 (3.44 mmol), N-bromosuccinimide (5.15 mmol) and2,2′-Azobis(2-methylpropionitrile) (0.7 mmol) in CCl₄ (4.0 mL) washeated to reflux for 4 h. The reaction mixture was cooled down and thesolid was filtered off. The filtrate was concentrated and the residuewas purified by flash chromatography (hexanes: AcOEt 2:1) to give thedesired product 58. MS ESI (m/z) 315 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ8.28 (dd, 1H, J₁=1.2, J₂=8.0 Hz), 8.09 (d, 1H, J=8.0 Hz), 7.86-7.82 (m,1H), 7.73-7.68 (m, 1H), 4.80 (s, 2H), 4.08 (s, 3H).

9-chloro-2-ethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one 59

To a solution of 58 (0.26 mmol) in ethanol (1 mL) was added EtNH₂ (2M inTHF, 0.76 mmol). The reaction mixture was heated to reflux for 2 h. Thesolvent was evaporated off and the residue was purified by flashchromatography (Hexanes: AcOEt 3:7) to give the desired product 59. MSESI (m/z) 247 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (d, 1H, J=8.4 Hz),8.13 (d, 1H, J=8.4 Hz), 7.87 (m, 1H), 7.72 (m, 1H), 4.53 (s, 2H), 3.77(q, 2H, J=7.2 Hz), 1.34 (t, 3H, J=7.2 Hz).

9-[(3-chloro-4-methoxybenzyl)amino]-2-ethyl-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-oneO

A solution of 59 (0.22 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.33 mmol) and (^(i)pr)₂NEt (1.31 mmol) in n-propanol(2.0 mL) was heated to 90° C. for 2 h. After cooling down, the resultingprecipitate O was collected by filtration. MS ESI (m/z) 382 (M+H); ¹HNMR (400 MHz, CDCl₃) δ 8.26 (t, 1H, J=6.0 Hz), 8.14 (dd, 1H, J₁=1.2,J₂=8.0 Hz), 7.94 (dd, 1H, J₁=1.2, J₂=8.8 Hz), 7.68-7.64 (m, 1H), 7.45(d, 1H, J=2.4 Hz), 7.33-7.29 (m, 1H), 6.95 (d, 1H, J=8.4 Hz), 4.98 (d,2H, J=5.6 Hz), 4.39 (s, 2H), 3.90 (s, 3H), 3.65 (q, 2H, J=7.6 Hz), 1.29(t, 3H, J=7.6 Hz).

Example 222-(tert-butyl)-9-[(3-chloro-4-methoxybenzyl)amino]-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one

2-(tert-butyl)-9-chloro-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one 60

To a solution of 58 (0.32 mmol) in ethanol (2.0 mL) was addedtert-butylamine (2M in THF, 0.95 mmol). The reaction mixture was heatedto reflux for 2 h. The solvent was evaporated off and the residue waspurified by flash chromatography (Hexanes: AcOEt 3:7) to give thedesired product 60. ¹H NMR (400 MHz, CDCl₃) δ 8.46 (dd, 1H, J₁=1.2,J₂=8.4 Hz), 8.12 (d, 1H, J=8.8 Hz), 7.88-7.84 (m, 1H), 7.73-7.69 (m,1H), 4.60 (s, 2H), 1.63 (s, 9H).

2-(tert-butyl)-9-[(3-chloro-4-methoxybenzyl)amino]-2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-oneP

A solution of 60 (0.07 mmol), 3-chloro-4-methoxybenzylaminehydrochloride (0.11 mmol) and (^(i)pr)₂NEt (0.44 mmol) in n-propanol(1.0 mL) was heated to 90° C. for 2 h. After cooling down, the resultingprecipitate P was collected by filtration. MS ESI (m/z) 410 (M+H)⁺; ¹HNMR (400 MHz, CDCl₃) δ 8.53 (t, 1H, J=6.4 Hz), 8.10 (d, 1H, J=8.4 Hz),7.92 (d, 1H, J=8.0 Hz), 7.63 (t, 1H, J=7.6 Hz), 7.45 (d, 1H, J=2.0 Hz),7.33-7.25 (m, 2H), 6.94 (d, 1H, J=8.8 Hz), 4.95 (d, 2H, J=6.8 Hz), 4.46(s, 2H), 1.57 (s, 9H).

Compounds exhibited PDE5 inhibition in the nanomolar range or below.Exemplary inhibition of representative compounds is shown in Table 3.

TABLE 3 PDE5 Inhibition of Representative Compounds. PDE5 EntryStructure Cmpd IC₅₀ (nM) 11

K 0.059 12

L 3.8 13

M 0.29 14

N 1.7 15

0 64.0 16

P 333.0

Example 23

A mixture of 26 (0.35 mmol), 3-chloro-4-(trifluoromethoxy)benzyl amine(1.05 mmol), TEA (1.05 mmol) and NaI (0.035 mmol) in NMP (2 mL) washeated to 130° C. and stirred for 24 h. The mixture was diluted withAcOEt (10 mL) and washed with H₂O (2×10 mL). The organic layers weredried over Na₂SO₄, filtered and evaporated under reduce pressure. FlashChromatography (eluent: 5% MeOH in AcOEt) gave the desired product Q(37% yield). MS ESI (m/z) 475 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s,1H), 8.07 (s, 1H), 7.77 (dd, 1H, J₁=1.6, J₂=8.4 Hz), 7.49 (d, 1H, J=2.0Hz), 7.35 (dd, 1H, J₁=0.8, J₂=8.4 Hz), 7.30 (dd, 1H, J₁=2.4, J₂=8.4 Hz),4.75 (s, 5H), 3.83 (t, 2H, J=6.4 Hz), 3.27 (s, 2H), 2.2 (s, 3H).

Example 24

A mixture of 26 (0.28 mmol), 3-chloro-4-fluorobenzyl amine (0.84 mmol),TEA (0.84 mmol) and NaI (0.028 mmol) in NMP (2 mL) was heated to 130° C.and stirred for 24 h. The mixture was diluted with AcOEt (10 mL) andwashed with H₂O (2×10 mL). The organic layers were dried over Na₂SO₄,filtered and evaporated under reduce pressure. Flash Chromatography(eluent: 5% MeOH in AcOEt) gave the desired product R (18% yield). MSESI (m/z) 409 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 8.03 (s,1H), 7.74-7.71 (m, 1H), 7.36 (dd, 1H, J₁=2.4, J₂=7.2 Hz), 7.20-7.12 (m,2H), 4.70 (s, 4H), 4.63 (s, 1H), 3.79 (t, 2H, J=6 Hz), 3.24 (s, 2H),2.18 (s, 3H).

Example 25

A mixture of 26 (0.27 mmol), 3-chloro-4-methoxyaniline (0.82 mmol), TEA(0.82 mmol) and NaI (0.027 mmol) in NMP (2 mL) was heated to 130° C. andstirred overnight. The mixture was diluted with AcOEt (10 mL) and washedwith H₂O (2×10 mL). The organic layers were dried over Na₂SO₄, filteredand evaporated under reduce pressure. Flash Chromatography (eluent: 5%MeOH in AcOEt) gave the desired product S (14% yield). MS ESI (m/z) 407(M+H)⁺.

Example 26

A mixture of 26 (0.25 mmol), 3-chloro-4-methoxyaniline (0.75 mmol), TEA(0.75 mmol) and NaI (0.025 mmol) in NMP (2 mL) was heated to 130° C. andstirred overnight. The mixture was diluted with AcOEt (10 mL) and washedwith H₂O (2×10 mL). The organic layers were dried over Na₂SO₄, filteredand evaporated under reduce pressure. Flash Chromatography (eluent: 5%MeOH in AcOEt) gave the desired product T (10% yield). MS ESI (m/z) 407(M+H)⁺.

Example 27

LiAlD₄ was added slowly to a solution of 65 in THF (2 mL) at 0° C. Thesolution was heated to 70° C. for 2 h. The reaction mixture was quenchedby adding NaOH 1M at 0° C. The aqueous phase was extracted with AcOEt(3×10 mL), dried over Na₂SO₄, filtered and evaporated under reducepressure. The residue was treated with HCl 2M in diethyl ether and theresulting precipitate (66) was collected by filtration (80% yield). 1HNMR (400 MHz, DMSO-d₆) δ 8.37 (s, 3H), 7.61 (d, 1H, J=2.4 Hz), 7.45-7.42(m, 1H), 7.18 (d, 1H, J=8.8 Hz), 3.95 (s, 2H), 3.86 (s, 3H).

A mixture of 26 (0.07 mmol), 66 (0.21 mmol), TEA (0.21 mmol) and NaI(0.007 mmol) in NMP (1 mL) was heated to 130° C. and stirred overnight.The mixture was diluted with AcOEt (10 mL) and washed with H₂O (2×10mL). The organic layers were dried over Na₂SO₄, filtered and evaporatedunder reduce pressure. Flash Chromatography (eluent: 2% MeOH in DCM)gave the desired product U (12% yield). MS ESI (m/z) 423 (M+H)⁺; ¹H NMR(400 MHz, CDCl₃) δ 8.35 (s, 1H), 7.97 (d, 1H, J=8.8 Hz), 7.74 (dd, 1H,J₁=1.8, J₂=9.0 Hz), 7.31 (d, 1H, J=2.0 Hz), 7.21 (dd, 1H, J₁=2.0, J₂=8.4Hz), 6.94 (d, 1H, J=8.0 Hz), 4.71 (s, 2H, CH₂), 4.56 (s, 1H, NH), 3.92(s, 3H, OCH₃), 3.82 (t, 2H, J=6.4 Hz, CH₂), 3.21 (t, 2H, J=6.4 Hz, CH₂),2.22 (s, 3H, COCH₃).

Substitution of H with D as shown in compound U led to the formation ofonly 1 metabolite instead of three.

Example 28

NaBH₃CN (10.52 mmol) was added to a solution of 67 (5.26 mmol) andNH₄OAc (52.6 mmol) in MeOH (15 mL). The reaction mixture was stirredovernight at 60° C. and then cooled to room temperature and partitionedbetween AcOEt (50 mL) and H₂O (50 mL). The aqueous layer was discardedand the organic phase was washed with water (2×50 mL), dried overNa₂SO₄, filtered and evaporated under reduce pressure to yield compound68 (61%). ¹H NMR (400 MHz, CDCl₃) δ 7.37 (d, 1H, J=2.0 Hz), 7.20 (dd,1H, J₁=2.0, J₂=8.4 Hz), 6.88 (d, 1H, J=8.0 Hz), 4.07 (q, 1H, J=6.4 Hz,CHCH₃), 3.88 (s, 3H, OCH₃), 1.84 (br s, 2H, NH₂), 1.36 (d, 3H, J=6.4 Hz,CHCH₃).

A mixture of 26 (0.175 mmol), 68 (0.526 mmol), TEA (0.7 mmol) and NaI(0.017 mmol) in NMP (1 mL) was heated to 130° C. and stirred overnight.The mixture was cooled down and AcOEt (10 mL) was added. The organiclayer was washed with H₂O (2×10 mL), dried over Na₂SO₄, filtered andevaporated under reduce pressure. Flash Chromatography (eluent: 2% MeOHin DCM) gave the desired product V (17% yield). MS ESI (m/z) 435 (M+H)⁺;¹H NMR (400 MHz, CDCl₃) δ 8.29 (S, 1H), 7.94 (d, 1H, J=8.4 Hz), 7.72 (d,1H, J=8.8 Hz), 7.25 (s, 1H), 7.16 (d, 1H, J=8.8 Hz), 6.88 (d, 1H, J=8.4Hz), 4.86 (t, 1H, J=6.8 Hz, NH), 4.71 (s, 2H, CH₂), 3.88 (s, 3H, OCH₃),3.84-3.72 (m, 3H, CH₂ and CH), 3.18 (t, 2H, J=5.6 Hz, CH₂), 2.22 (s, 3H,COCH₃), 1.68 (d, 3H, J=5.2 Hz, CHCH₃).

Example 29

A mixture of 26 (0.175 mmol), 69 (0.526 mmol), TEA (0.7 mmol) and NaI(0.017 mmol) in NMP (1 mL) was heated to 130° C. and stirred overnight.The mixture was cooled down and AcOEt (10 mL) was added. The organiclayer was washed with H₂O (2×10 mL), dried over Na₂SO₄, filtered andevaporated under reduce pressure. Flash Chromatography (eluent: 2% MeOHin DCM) gave the desired product W (21% yield). MS ESI (m/z) 435 (M+H)⁺;¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 8.01 (s, 1H), 7.73 (d, 1H, J=8.8Hz), 7.17 (s, 1H), 7.11 (dd, 1H, J₁=2.0, J₂=8.4 Hz), 6.92 (d, 1H, J=9.0Hz), 4.62 (s, 2H), 3.91-3.85 (m, 6H), 3.81 (t, 2H, J=6.0 Hz), 3.23 (s,2H), 2.95 (t, 2H, J=6.0 Hz), 2.22 (s, 3H).

Compounds exhibited PDE5 inhibition in the nanomolar range or below.Exemplary inhibition of representative compounds is shown in Table 4.

TABLE 4 PDE5 Inhibition of Representative Compounds. R³ R⁸ R⁵ A X V W mn R² R¹ cmpd PDE5 IC₅₀ (nM) CN H Ac NH CH₂ bond bond 1 2 OCF₃ Cl Q 337CN H Ac NH CH₂ bond bond 1 2 F Cl R 1.5 CN H Ac NH bond bond bond 1 2OMe Cl S 593 CN H Ac NH bond bond bond 1 2 Cl OMe T 425 CN H Ac NH CD₂bond bond 1 2 OMe Cl U 0.044 CN H Ac NH CHMe bond bond 1 2 OMe Cl V 26.9CN H Ac NH (CH₂)₂ bond bond 1 2 OMe Cl W 10.6

Benzo[b][1,6]naphthyridine derivatives—Formula Ia Example 30

Example 31

Example 32

TABLE 5 PDE5 Inhibition of Representative Compounds-Formula Ia.

PDE5 IC₅₀ R³ R⁸ R⁵ A X V W m n R² R¹ Z R cmpd (nM) CN H Ac NH CH₂ bondbond 1 2 — Cl N H AA 57 CN H Ac NH CH₂ bond bond 1 2 F F C F AB 3.3 CN HAc NH CH₂ bond bond 1 2 OMe Cl C F AC 0.32

2,3-dihydro-1H-pyrrolo[3,4-b]quinolin-1-one derivatives, with deuterium(D) Example 33

R³ R⁸ R⁵ A X V W m n R² R¹ cmpd CN H Et NH CD₂ C(O) bond 0 1 OMe Cl AD

The following prophetic examples are provided to illustrate otheraspects of the present invention:

R³ R⁵ R¹⁵ R¹⁶ B A X V W m n R² R¹ cmpd CN Ac H — N NH CD₂ bond bond 1 2OMe Cl BA CN Ac H F C NH CD₂ bond bond 1 2 OMe Cl BB CN Ac F H C NH CD₂bond bond 1 2 OMe Cl BC CN Ac H OH C NH CD₂ bond bond 1 2 OMe Cl BD CNAc OH H C NH CD₂ bond bond 1 2 OMe Cl BE CN Ac H OMe C NH CD₂ bond bond1 2 OMe Cl BF CN Ac OMe H C NH CD₂ bond bond 1 2 OMe Cl BG

Example 34

Example 35

Example 36

Example 37

Example 38

Although the invention has been described and illustrated in theforegoing illustrative embodiments, it is understood that the presentdisclosure has been made only by way of example, and that numerouschanges in the details of implementation of the invention can be madewithout departing from the spirit and scope of the invention, which islimited only by the claims that follow. Features of the disclosedembodiments can be combined and/or rearranged in various ways within thescope and spirit of the invention to produce further embodiments thatare also within the scope of the invention. Those skilled in the artwill recognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific embodimentsdescribed specifically in this disclosure. Such equivalents are intendedto be encompassed in the scope of the following claims.

1. A compound of formula (I),

wherein A is O or NR₄; B is CR¹⁶ or N; V is a bond or C(O); W is a bondor NR¹³; X is —(C₁-C₃)-alkyl, —(C1

C3)-alkyl substituted with at least one D, C(O), S, S(O), or S(O)₂; Y isNR⁵, O or S; Z is C or N; R¹ is hydrogen, halogen or —(C₁-C₆)-haloalkyl;R² is hydrogen or —OR⁶; R³ is hydrogen, —OMe, —CF₃, —CN or halogen; R⁴is hydrogen or —(C₁-C₃)-alkyl; R⁵ is hydrogen, —(C₁-C₃)-alkyl,—(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷, —C(O)N(R⁷)₂, —S(O)₂R⁷; R⁶ ishydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or —(C₃-C₈)-cycloalkyl; R⁷is independently hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, or aryl;R⁸ is hydrogen, —(C₁-C₆)-alkyl, —(C₁-C₆)-haloalkyl, —(C₃-C₈)-cycloalkyl,—NR⁹R1, —S(O)₂R¹¹, or heterocyclyl; R⁹ and R¹⁰ are each independentlyhydrogen, —(C₁-C₆)-alkyl, —(C₃-C₈)-cycloalkyl, or —C(O)R¹¹, wherein the—(C₁-C₆)-alkyl or —(C₃-C₈)-cycloalkyl are optionally substituted with—(C₁-C₆)-alkyl, —(C₃-C₈)-cycloalkyl, —NR¹¹R¹², —SR¹¹, or heterocyclyl;or, R⁹ and R¹⁰ together with the nitrogen atom to which they areattached form a 3 to 8-membered heterocycle, wherein any one of the ringcarbon atoms is optionally replaced with a heteroatom, and wherein theheterocycle is optionally substituted with —(C₁-C₆)-alkyl; R¹¹ and R¹²are each independently hydrogen, —(C₁-C₆)-alkyl, or —(C₃-C₈)-cycloalkyl;R¹³ is hydrogen, —(C₁-C₆)-alkyl, —(C₃-C₅)-cycloalkyl, —C(O)R⁷, —C(O)OR⁷,—C(O)N(R⁷)₂, or —S(O)₂R⁷; R¹⁴ is hydrogen, halogen or—(C₁-C₆)-haloalkyl; R¹⁵ is hydrogen, —OR¹⁷, —OH or halogen; R¹⁶ ishydrogen, —OR¹⁷, —OH or halogen; R¹⁷ hydrogen, —(C₁-C₆)-alkyl,—(C₁-C₆)-haloalkyl, or —(C₃-C₈)-cycloalkyl; m and n are each 0, 1, 2, or3, provided that the sum of m+n is an integer from 1-4; or apharmaceutically acceptable salt or tautomer thereof; with the provisothat R¹ and R² are not both hydrogen when V and W are each a bond, Y isNR⁵, A is NR⁴, X is CO, n=2 and m=1.
 2. The compound of claim 1, havingformula (Ia):


3. The compound of claim 1, having formula (Ib):


4. The compound of claim 1, having formula (Ic):


5. The compound of claim 1, wherein X is —(C₁-C₃)-alkyl.
 6. The compoundof claim 1, wherein X is —(C₁-C₃)-alkyl substituted with at least one D.7. The compound of claim 1, wherein A is NR⁴; V is a bond or C(O); W isa bond or NR¹³; X is —(C₁-C₃)-alkyl or —(C₁-C₃)-alkyl substituted withat least one D; Y is NR⁵; R¹ is halogen or —(C₁-C₃)-haloalkyl; R² is—OR⁶; R³ is hydrogen, —OMe, —CF₃, —CN or halogen; R⁵ is hydrogen,—(C₁-C₃)-alkyl, —(C₁-C₃)-cycloalkyl, or —C(O)R⁷; R⁶ is —(C₁-C₃)-alkyl or—(C₁-C₃)-haloalkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen or—(C₁-C₃)-alkyl; R¹³ is hydrogen or —(C₁-C₃)-alkyl; and m and n areindependently 0, 1 or 2, provided that the sum of m+n is an integer from2-3.
 8. The compound of claim 1, wherein A is NH; V is a bond or C(O); Wis a bond or NR¹³; X is —(C₁-C₃)-alkyl or —(C₁-C₃)-alkyl substitutedwith at least one D; Y is NR⁵; R¹ is halogen; R² is —OR⁶; R³ ishydrogen, —OMe, —CF₃, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or—C(O)R⁷; R⁶ is —(C₁-C₃)-alkyl; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen or—(C₁-C₃)-alkyl; R¹³ is hydrogen; m is 0 or 1; and n is
 2. 9. Thecompound of claim 1, wherein A is NH; V is a bond or C(O); W is a bondor NH; X is —CH₂— or —CD₂; Y is NR⁵; R¹ is halogen; R² is —OCH₃; R³ ishydrogen, —OMe, —CF₃, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or—C(O)CH₃; R⁸ is hydrogen or —(C₁-C₃)-alkyl; m is 0 or 1; and n is
 2. 10.The compound of claim 1, wherein V and W are each a bond, or V is C(O)and W is NR¹³; X is —(C₁-C₃)-alkyl or —(C₁-C₃)-alkyl substituted with atleast one D; Y is NR⁵; and m and n are each 0, 1 or 2, provided that thesum of m+n is an integer from 2-3.
 11. The compound of claim 1, whereinA is NH; V is C(O); W is NH; X is CH₂ or CD₂; Y is NR⁵; R¹ is halogen or—(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ is hydrogen, —OMe, —CF₃, —CN orhalogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, or C(O)R⁷; R⁶ is hydrogen,—(C₁-C₃)-alkyl, or —(C₁-C₃)-haloalkyl; R⁷ is hydrogen or —(C₁-C₃)-alkyl;R⁸ is hydrogen, —(C₁-C₃)-alkyl, —(C₁-C₃)-haloalkyl, —(C₃-C₅)-cycloalkyl,—NR⁹R¹⁰; m is 0 or 1; and n is
 2. 12. The compound of claim 1, wherein Ais NH; V is C(O); W is NH; X is CH₂ or CD₂; Y is NR⁵; R¹ is halogen; R²is —OCH₃; R³ is hydrogen, —OMe, —CF₃, —CN or halogen; R⁵ is hydrogen,—(C₁-C₃)-alkyl, or C(O)R⁷; R⁷ is —(C₁-C₃)-alkyl; R⁸ is hydrogen or—(C₁-C₃)-alkyl; m is 0; and n is
 2. 13. The compound of claim 1, whereinA is NH; V and W are each a bond, or V is C(O) and W is NH; X is CH₂ orCD₂; Y is NR⁵; R¹ is halogen or —(C₁-C₃)-haloalkyl; R² is —OR⁶; R³ ishydrogen, —OMe, —CF₃, —CN or halogen; R⁵ is hydrogen, —(C₁-C₃)-alkyl, orC(O)R⁷; R⁶ is hydrogen, —(C₁-C₂)-alkyl, or —(C₁-C₂)-haloalkyl; R⁷ is—(C₁-C₃)-alkyl; R⁸ is hydrogen, —(C₁-C₃)-alkyl, —(C₁-C₃)-haloalkyl, or—(C₃-C₅)-cycloalkyl; m is 0 or 1; and n is
 2. 14. The compound of claim1, wherein A is NH; V and W are each a bond; X is CH₂ or CD₂; Y is NR⁵;R¹ is chlorine; R² is —OCH₃; R³ is —CN; R⁵ is hydrogen or—(C₁-C₃)-alkyl; R⁸ is hydrogen or —(C₁-C₃)-alkyl; m is 1; and n is 2.15. The compound of claim 1, wherein A is NH; V and W are each a bond; Xis CH₂ or CD₂; Y is NC(O)CH₃; R¹ is halogen; R² is —OCH₃; R³ ishydrogen, —OMe, —CF₃, —CN or halogen; R⁸ is hydrogen; m is 1; and n is2.
 16. A composition comprising a compound claim 1 and apharmaceutically acceptable carrier.
 17. A method of treatingneurodegenerative disease in a subject comprising administration of atherapeutically effective amount of a compound of claim
 1. 18. Themethod of claim 17, wherein the disease is Alzheimer's Disease.
 19. Amethod of increasing long-term potentiation in a subject comprisingadministration of a therapeutically effective amount of a compound ofclaim
 1. 20. The method of claim 19, wherein the disease is Alzheimer'sDisease.
 21. A method of improving memory in a subject comprisingadministration of a therapeutically effective amount of a compound ofclaim
 1. 22. The method of claim 21, wherein the subject has aneurodegenerative disease.
 23. The method of claim 22, wherein theneurodegenerative disease is Alzheimer's Disease.
 24. (canceled)